Methods of suppressing microglial activation and systemic inflammatory responses

ABSTRACT

Methods of suppressing the activation of microglial cells in the Central Nervous System (CNS), methods of ameliorating or treating the neurological effects of cerebral ischemia or cerebral inflammation, and methods of combating specific diseases that affect the CNS by administering a compound that binds to microglial receptors and prevents or reduces microglial activation are described. ApoE receptor binding peptides that may be used in the methods of the invention are also described, as are methods of using such peptides to treat peripheral inflammatory conditions such as sepsis. Also described are methods of screening compounds for the ability to suppress or reduce microglial activation.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. patent application Ser. No.10/252,120, filed Sep. 23, 2002, now abandoned, the disclosure of whichis incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under NIH grantsNS368087-01A2, K08NS01949, and RO3AG16507-01. The Government has certainrights to this invention.

FIELD OF THE INVENTION

The present invention relates to methods of suppressing the activationof microglial cells in the Central Nervous System (CNS), methods ofreducing or suppressing the activation of glial or microglial cells,methods of ameliorating or treating the neurological effects of cerebralischemia or cerebral inflammation, methods of combating specificdiseases that affect the CNS by administering a compound that binds tomicroglial receptors and prevents or reduces microglial activation, andmethods of screening compounds for the ability to prevent or reducemicroglial activation. The invention further relates to the methods ofsuppressing glutamate excitotoxicity and neuronal cell death associatedwith N-methyl-D-aspartate (NMDA) exposure, as well as methods forsuppressing systemic inflammatory responses such as those seen insepsis.

BACKGROUND OF THE INVENTION

The Central Nervous System (CNS) has long been considered to be a siteof relative immune privilege. However, it is increasingly recognizedthat CNS tissue injury in acute and chronic neurological disease may bemediated by the CNS inflammatory response. The CNS inflammatory responseis primarily mediated by inflammatory cytokines.

The microglial cell is the primary immunocompetent cell in the centralnervous system. Microglia are morphologically, immunophenotypically andfunctionally related to cells of the monocyte/macrophage lineage(Gehrmenn et al., 1995). Acute CNS insult, as well as chronic conditionssuch as HIV encephalopathy, epilepsy, and Alzheimer's disease (AD) areassociated with microglial activation (McGeer et al., 1993; Rothwell andRelton, 1993; Giulian et al., 1996; Sheng et al., 1994). Microglialactivation results in the production of nitric oxide (NO) and other freeradical species, and the release of proteases, inflammatory cytokines(including IL-1β, IL-6 and TNFα), and a neurotoxin that works throughthe NMDA receptor (Giulian et al., 1996). Microglial activation can beassessed by measuring the production of nitrite, a stable product ofnitric oxide formation (Barger and Harmon, 1997).

Apolipoprotein E (ApoE) plays a role in cholesterol metabolism and hasalso been reported to have immunomodulatory properties. For instance,ApoE has been demonstrated to have immunomodulatory effects in vitro,including suppression of lymphocyte proliferation and immunoglobulinsynthesis after mitogenic challenge (Avila et al., 1982; Edgington andCurtiss, 1981). ApoE is secreted in large quantities by macrophage afterperipheral nerve injury, and by astrocytes and oligodendrocytes (glialcells) after CNS injury (Stoll et al., 1989; Stoll and Mueller, 1986).The role that ApoE plays in glial activation and CNS injury, however,remains controversial.

The majority of ApoE is produced in the liver. However, due to its largesize, ApoE does not readily cross the blood-brain barrier. In fact,following liver transplantation, peripheral apoE phenotype changes tothat of the donor liver, while CSF (cerebrospinal fluid) apoE phenotyperemains unchanged (Linton et al., 1991). Thus, ApoE localized within thenervous system represents a discrete pool from protein produced in theperiphery (Laskowitz et al., January 2001).

ApoE is a 299 amino acid lipid-carrying protein with a known sequence(Rail et al., J. Biol. Chem. 257:4174 (1982); McLean et al., J. Biol.Chem. 259:6498 (1984)). The complete gene for human ApoE has also beensequenced (Paik et al., Proc. Natl. Acad. Sci. USA 82:3445 (1985). ApoEsequences from at least ten species have been determined, and show ahigh degree of conservations across species, except at the amino andcarboxyl termini. Weisgraber, Advances in Protein Chemistry 45:249(1994).

Human ApoE is found in three major isoforms: ApoE2, ApoE3, and ApoE4;these isoforms differ by amino acid substitutions at positions 112 and158. The most common isoform is ApoE3, which contains cysteine atresidue 112 and arginine at residue 158; ApoE2 is the least commonisoform and contains cysteine at residues 112 and 158; ApoE4 containsarginine at residues 112 and 158. Additional rare sequence mutations ofhuman ApoE are known (see, e.g., Weisgraber, Advances in ProteinChemistry 45:249 (1994), at page 268-269).

ApoE has two distinct functional domains, a 10-kDa carboxyl terminus anda 22 k-Da amino terminus (Wetterau et al., 1988). The carboxyl terminushas a high affinity for lipid and mediates the role of ApoE incholesterol transport. The amino terminus is composed of fourantiparallel alpha helices, which includes the receptor binding region(Weisbarger et al., 1983; Innerarity et al., 1983). ApoE is known tobind a family of cell surface receptors, including the LDL, VLDL,LRP/α2M, ER-2, LR8 receptors, apoE receptor 2 (apoER2), andmegalin/gp330 (Kim et al., 1996; Novak et al., 1996; Veinbergs et al.,2001). The interaction of apolipoprotein E and the LDL receptor isimportant in lipoprotein metabolism. In studies of the LDLreceptor-binding activity of ApoE, it is typically complexed withphospholipid. The protein has been described as essentially inactive inthe lipid-free state (Innerarity et al., 1979).

One region of ApoE which is critical for the interaction with the LDLreceptor lies between residues 140-160 (Mahley, 1988), and site-specificmutagenesis studies of this region have demonstrated that mutationsaffecting charge and conformation can result in defective binding(Lalazar, 1988). The receptor binding region of ApoE (i.e., amino acidresidues 135-160) is rich in basic amino acids including arginine andlysine. Various amino acid substitutions in the receptor binding regionof ApoE have been studied for their effects on ApoE-LDL receptorbinding. Substitution of either arginine or lysine at residues 136, 142,145 and 146 with neutral residues decreased normal ApoE3 bindingactivity (Weisgraber, 1988). No single substitution of a basic residuewithin the receptor-binding region of ApoE3 completely disrupts LDLreceptor binding, suggesting that no one residue is critical for thisinteraction. It has been postulated that regions of ApoE outside the LDLbinding region are necessary to maintain the receptor-binding region inan active binding conformation (Weisgraber, 1994). Dyer et al. (1991)studied lipid-free synthetic peptide fragments comprising residues141-155 of ApoE, and a dimeric peptide of this sequence. No bindingactivity was observed with the monomer of this peptide, but low levelsof binding were observed with the dimer (˜1% of LDL activity).

The receptors that bind ApoE have areas of high sequence similarity. Thescavenger receptor is known to be present on microglia, andpreferentially binds acetylated and oxidized LDL. The scavenger receptormay be particularly relevant under inflammatory (oxidizing) conditions.Scavenger receptors are also known to be upregulated in microglia afterinjury (Bell et al., 1994).

LRP receptors are known to be present on macrophages. In overview,following modification by lipoprotein lipase and the association ofapolipoproteins, very large density lipoproteins (VLDL) and chylomicronbecome remnants, and are cleared hepatically by a receptor-mediatedmechanism. Although recognized as distinct from the low densitylipoprotein (LDL) receptor, the remnant receptor also has a highaffinity for ApoE, and recognizes the remnant particles via incorporatedApoE moieties. In 1988, this remnant receptor was cloned, and dubbed theLDL receptor-related related protein, or “LRP”.

The LRP is a large receptor, with a primary sequence of 4525 aminoacids, and bears many structural similarities to other members of theLDL receptor family. Like the LDL receptor, the extracellular domain ofLRP includes a cysteine-enriched ligand binding domain and EGF precursorhomology domain which are believed to play a role in the acid-dependentdissociation of ligand from the receptor. Unlike the LDL receptor,however, the O-lined sugar domain is not present in the extracellularportion adjacent to the membrane. As with all of the members of the LDLreceptor family, LRP is a transmembrane protein, and is anchored by asingle transmembrane segment. The cytoplasmic tail of the protein is 100amino acids, approximately twice as long as the LDL receptor, andcontains the NpxY motif, which is believed to be necessary for targetedcoated-pit mediated endocytosis (Krieger and Herz, 1994; Misra et al.,1994).

In addition to binding lipid, ApoE also binds lipopolysaccharide (LPS),which is an endotoxin that mediates Gram-negative sepsis by inducing theproduction of macrophage-derived cytokines. These cytokines, whichinclude TNFα, IL-1α, IL-1β and IL-6, are responsible for the metabolicand physiologic changes that ultimately lead to pathology (Waage et al.,1987; Chensue et al., 1991: Henderson et al., 1996). ApoE redirectsbound LPS from macrophages to parenchymal liver cells, which mediate thesubsequent secretion of LPS into the bile where it is inactivated(Harris et al., 1993; Harris et al., 1998). Consequently, macrophagesbecome less activated and produce less of the proinflammatory mediators.

Laskowitz et al. (June 1997) described experiments in which mixedneuronal-glial cell cultures from apoE-deficient mice were stimulatedwith lipopolysaccharide (LPS). It was found that preincubation of thecell cultures with apoE blocked glial secretion of TNFα in adose-dependent manner. More recently, Van Oosten et al. demonstratedthat concomitant administration of ApoE with a lethal dose of LPSprotected mice against LPS-induced mortality (Van Oosten et al., 2001).Rensen et al. demonstrated that a free ApoE molecule binds approximatelytwo molecules of LPS, possibly by an exposed hydrophilic domaininvolving arginine residues since selective elimination of the positivecharge on arginine residues of apoE resulted in a largely reducedbinding of LPS to ApoE and abolished the effect of ApoE on the in vivobehavior of LPS (Rensen et al., 1997). Interestingly, lactoferrin is aglycoprotein with an arginine/lysine-rich sequence at positions 25-31resembling the receptor binding site (amino acids 142-148) of ApoE, andhas also been shown to bind LPS (Huettinger et al., 1988; Cohen et al.,1992; Miyazawa et al., 1991). Although, he showing by Laskowitz et al.that preincubation of ApoE with neuronal-glial cell cultures blockedLPS-induced TNF-alpha tion whereas coadministration of ApoE with LPS didnot suggests that some other mechanism than LPS binding is involved(Laskowitz et al., June 1997).

In addition to its roles in cholesterol metabolism and endotoxinclearance, ApoE may also play an important role in neurological disease.The presence of ApoE4 has been associated with risk of developingsporadic and late-onset Alzheimer's disease (Strittmatter et al., 1993).Barger and Harmon (August 1997) reported that treatment of microgliawith a secreted derivative of beta-amyloid precursor protein(sAPP-alpha) activated microglia, induced inflammatory reactions inmicroglia, and enhanced the production of neurotoxins by microglia. Theability of sAPP-alpha to activate microglia was blocked by priorincubation of the sAPP-alpha protein with apolipoprotein E3 but notapolipoprotein E4. More recently, some researchers have proposed aninvolvement of ApoE in regulating Tau phosphorylation, suggesting thatApoE is involved some way in the development of the neurofibrillaryfibrils associated with Alzheimer's Disease (Flaherty et al., 1999;Tesseur et al., 2000). However, the link between ApoE and Tau hasremained controversial (Lovestone, 2001).

There have also been numerous clinical and experimental observationsdemonstrating that ApoE modifies the response of brain to acute injury.For example, clinical observations suggest that the ApoE4 allele isassociated with increased mortality and functional deficit after acuteand chronic closed head injury (Sorbi et al., 1995; Teasdale et al.,1997; Jordan et al., 1997; Friedman et al., 1999). The ApoE4 allele hasalso been associated with the extent of amyloid β-protein depositionfollowing head injury (Mayeux et al., 1995; Nicoll et al., 1995).

The deleterious effects of the apoE4 isoform on neurological outcomeshave also been observed in a variety of clinical settings associatedwith cerebral ischemia. These include stroke (Slooter et al., 1997),intracranial hemorrhage (Alberts et al., 1995; McCarron et al., 1998),cognitive deficit after cardiopulmonary bypass (Tardiff et al., 1997)and hypoxic brain injury following cardiac arrest resuscitation(Schiefermeier et al., 2000). The role of ApoE following focal ischemiais less clear, however, with at least one clinical study failing todocument an effect of apoE genotype on functional outcome followingstroke (Broderick et al., 2001).

Clinical observations implicating a role for apoE in modifying thecentral nervous system response to ischemia have recently been extendedto animal models. ApoE deficient mice have larger infarcts and worsefunctional outcomes following focal ischemia and reperfusion relative tocontrol animals matched for age, sex, and genetic background (Laskowitzet al., July 1997).

This effect is independent of cerebral blood flow or cerebrovascularanatomy (Bart et al., 1998). In models of transient forebrain ischemia,apoE deficient animals also have increased injury to neuronalpopulations that are selectively vulnerable to cerebral hypoperfusion,including hippocampus, caudoputamen, and cortex (Sheng et al., 1999;Horsburgh et al., 1999). This increased sensitivity to ischemia can bereversed by intraventricular administration of human recombinant apoE(Horsburgh et al., 2000). Moreover, consistent with the clinicalliterature, apoE deficient mice expressing the human apoE4 transgenehave larger infarcts and worse functional outcomes than mice expressingthe human apoE3 transgene (Sheng et al., 1998).

Although there are multiple clinical reports demonstrating that apoEgenotype influences neurological recovery in isoform-specific fashion,the mechanisms by which this occur remain poorly defined. It has beenproposed that endogenous apoE may influence the CNS response to injuryby modifying oxidative stress (Miyata and Smith, 1996), exerting directneurotrophic effects (Holtzman et al., 1995), downregulating the CNSinflammatory response (Lynch et al., 2001), or serving as a pathologicalchaperone by promoting cerebral amyloid deposition (Wisniewski andFrangione, 1992). More recent studies, however, have failed todemonstrate any neuroprotective effect from the intact ApoE protein(Jordan et al., 1998; Lendon et al., 2000).

Furthermore, several studies have suggested that ApoE derived peptidefragments may cause neuronal injury. For example, it has recently beendemonstrated that carboxyl-terminal truncated forms of apoE occur in thebrains of patients with AD, presumably as a result of intracellularprocessing. These fragments are bioactive and are capable of interactingwith cytoskeletal proteins to induce inclusions resemblingneurofibrillary tangles in cultured neurons (Huang et al., 2001).Moulder et al. recently reported that a dimer composed of theApoE-derived peptide 141-155 has a neurotoxic effect, suggesting to theauthors that ApoE itself could be a source of toxicity in Alzheimer'sdisease brain (Moulder et al., 1999). Using a peptide comprised of atandem repeat of residues 141-149, Tolar et al. demonstrated thatexposure of primary hippocampal neurons to this peptide induced neuronalcell death, an effect which was blocked by preincubation with MK-801, anNMDA antagonist (Tolar et al., 1999). These results predict thatexposure with this tandem repeat peptide amplifies NMDA-inducedexcitotoxicity by direct or indirect mechanisms.

In summary, ApoE plays varied roles in different biological processes.While ApoE appears to provide a protective effect in the periphery byremoving LPS from macrophages, the role it plays in CNS injury andneurological diseases such as Alzheimer's Disease is far from clear.What is needed is a better understanding of how ApoE contributes to theCNS inflammatory response, to aide in the formulation of reagents foruse in the treatment of neurological injury and disease.

SUMMARY OF THE INVENTION

The present invention is based on the finding that microglial activationcan be reduced or suppressed using peptides that comprise the receptorbinding site sequence of Apolipoprotein E. Thus, the present inventionprovides methods and compositions for treating CNS disease states inwhich glial or microglial activation occurs, and in which glial ormicroglial activation contributes to the deleterious signs and/orsymptoms associated with the specific disease state.

The present invention is further based on the unexpected finding thatpeptides derived from the receptor-binding region of ApoE completelysuppress the neuronal cell death and calcium influx associated withN-methyl-D-aspartate exposure. This result is in contrast to recentreports in the literature that ApoE enhances the NMDA-inducedexcitotoxicity, a surprising result which provides the basis forApoE-based formulations and treatments for injury and diseasesassociated with activation of glutamate receptors such as the NMDAreceptor.

The present invention is further based on the unexpected finding thatpeptides derived from the receptor-binding region of ApoE protectagainst LPS-induced production of TNFα and IL-6 in an in vivo sepsismodel, a finding that is surprising in view of the fact that thereceptor-binding fragments of the present invention contain only a smallportion of the intact protein. Thus, the present invention providesmethods and compositions for treating sepsis using the peptides of thepresent invention, as well as any compound identified using the methodsdisclosed herein that binds to the same receptor as the peptides of thepresent invention.

The present invention is further based upon the identification andcharacterization of the LRP/α2M as a high affinity Apolipoprotein Ereceptor, a characterization which is the basis for several of thediagnostic assays and kits disclosed herein. The assays of the inventionmay be performed using any ApoE receptor, including but not limited tothe LRP/α2M, LDL, VLDL, ER-2, LR8, apoER₂ and megalin/gp 330 receptors.

In view of the foregoing, one aspect of the present invention is amethod of suppressing glial or microglial activation, either in vitro orin a mammalian subject, by administering a compound or compositioncontaining a compound that binds to glial or microglial cells or othereffector cells such as astrocytes at the receptor bound by the peptidesof the present invention, and particularly the peptides of SEQ ID Nos 3,4, 5, 6 and 10. The compound or composition is administered in an amountthat reduces glial or microglial activation compared to activation thatwould occur in the absence of the compound. Suitable compounds includethe peptides of the present invention, which may be formulated intopharmaceutical compositions comprising one or more of the peptides aloneor in combination with other pharmaceutical compounds relevant forsuppressing glial or microglial activation.

A further aspect of the present invention is a method of amelioratingsymptoms associated with CNS inflammation by administering a compound ora composition containing a compound that binds to glial or microglialcells at the receptor bound by the peptides of the present invention,and particularly the peptides of SEQ ID Nos 3, 4, 5, 6 and 10. Thecompound or composition is administered in an amount that reduces CNSinflammation as compared to that which would occur in the absence of thecompound. Suitable compounds include the peptides of the presentinvention, which may be formulated into pharmaceutical compositionscomprising one or more of the peptides alone or in combination withother pharmaceutical compounds relevant for suppressing CNSinflammation.

A further aspect of the present invention is a method of amelioratingsymptoms associated with CNS or cerebral ischemia in a subject, byadministering a compound or a composition containing a compound thatbinds to glial or microglial cells at the LRP/α2M receptor or at thereceptor bound by the peptides of the present invention, andparticularly the peptides of SEQ ID Nos 3, 4, 5, 6 and 10. The compoundor composition is administered in an amount that alleviates symptomsassociated with CNS ischemia as compared to that which would occur inthe absence of the compound. Suitable compounds include the peptides ofthe present invention, which may be formulated into pharmaceuticalcompositions comprising one or more of the peptides alone or incombination with other pharmaceutical compounds relevant for alleviatingCNS ischemia.

A further aspect of the present invention is a method of reducingneuronal cell death associated with glutamate excitotoxicity or NMDAexposure in a mammalian subject by administering to said subject acompound or a composition containing a compound that binds to glial ormicroglial cells at the receptor bound by the peptides of the presentinvention, and particularly the peptides of SEQ ID Nos 3, 6 and 10. Thecompound or composition is administered in an amount that reducesneuronal cell death associated with glutamate toxicity as compared toreduction that would occur in the absence of the compound. Suitablecompounds include the peptides of the present invention, which may beformulated into pharmaceutical compositions comprising one or more ofthe peptides alone or in combination with other pharmaceutical compoundsrelevant for suppressing glutamate toxicity.

A further aspect of the present invention is a method of suppressingmacrophage activation in a mammalian subject, by administering acompound or a composition containing a compound that binds to macrophagecells at the LRP/α2M receptor or at the receptor bound by the peptidesof the present invention, and particularly the peptides of SEQ ID Nos 3,6 and 10. The compound or composition is administered in an amount thatsuppresses macrophage activation as compared to activation that wouldoccur in the absence of the compound. Suitable compounds include thepeptides of the present invention, which may be formulated intopharmaceutical compositions comprising one or more of the peptides aloneor in combination with other pharmaceutical compounds relevant forsuppressing macrophage activation.

A further aspect of the present invention is a method of treatingatherosclerosis or of reducing the formation of atherosclerotic plaques,comprising administering a compound or a composition containing acompound that binds to macrophage cells at the receptor bound by thepeptides of the present invention, and particularly the peptides of SEQID Nos 3, 4, 5, 6 and 10. The compound or composition is administered inan amount that reduces the formation of artherosclerotic plaques ascompared to that which would occur in the absence of the compound.Suitable compounds include the peptides of the present invention, whichmay be formulated into pharmaceutical compositions comprising one ormore of the peptides alone or in combination with other pharmaceuticalcompounds relevant for reducing the formation of atheroscleroticplaques.

A further aspect of the present invention is a method of treating or ofreducing the inflammation associated with bacterial sepsis, comprisingadministering a compound or a composition containing a compound thatbinds to macrophage cells at the receptor bound by the peptides of thepresent invention, and particularly the peptides of SEQ ID Nos 3, 4, 5,6 and 10. The compound or composition is administered in an amount thatreduces sepsis-associated inflammation as compared to that which wouldoccur in the absence of the compound. Suitable compounds include thepeptides of the present invention, which may be formulated intopharmaceutical compositions comprising one or more of the peptides aloneor in combination with other pharmaceutical compounds relevant fortreating sepsis.

A further aspect of the present invention is a therapeutic peptide ofSEQ ID NO: 3, or a dimer of two peptides wherein each peptide comprisesSEQ ID NO:2, or a peptide of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or apeptide of SEQ ID NO:10, and pharmaceutical compositions thereof. Alsoincluded are compounds identified using the assays disclosed in thepresent invention, wherein such compounds bind to the receptor bound bythe peptides of the invention and mediate the functional effectsdisclosed herein. Consistent therewith, the invention also includes useof the disclosed peptides and compounds and functional variants thereofin methods of making medicaments for treating the various diseases anddisorders discussed herein. Such medicaments may comprise the subjectpeptides and compounds of the invention, alone or in combination withother known pharmaceutical agents.

A further aspect of the present invention is a method of screening acompound for the ability to suppress glial or microglial activation byincubating an activated glial or microglial cell culture with thecompound, and then measuring a marker of microglial activation such asnitric oxide.

A further aspect of the present invention is a method of screening acompound for the ability to suppress glial or microglial activation, bypre-incubating a glial or microglial cell culture with the compound;incubating the cell culture with a known activator of glia or microglia;and then measuring a marker of glial or microglial activation.

A further aspect of the present invention is a method of screening atest compound for the ability to suppress glial or microglialactivation, by determining whether the compound binds to glia ormicroglia at the same receptor to which peptides of SEQ ID NO:3 or SEQID No:4 or SEQ ID No:5 or SEQ ID NO:6 or SEQ ID NO:10 bind (forinstance, the LRP/α2M receptor).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphs the production of nitrite by cultures of glial cells fromApoE-deficient mice (solid bar), ApoE3 transgenic mice (hatched bar),and control mice (white bar), after exposure to lipopolysaccharide(LPS). Responses were measured at 24 and 60 hours after stimulation ofcell cultures by LPS.

FIG. 2 graphs nitrite production by enriched microglia primary culturesfrom ApoE-deficient mice after stimulation with LPS and subsequentaddition of peptides of SEQ ID NO:3 (tandem repeat peptides). Peptideswere added in doses of from 0 μM to 1000 μM, and a dose dependentdecrease in nitrite production was observed. As a control, peptides ofSEQ ID NO:2 were added to cultures (solid bar); no decrease in nitriteproduction was observed.

FIG. 3A graphs intracellular calcium content over time in murineperitoneal macrophages, after exposure to either ApoE3 (squares) orApoE4 (circles).

FIG. 3B graphs inositol trisphosphate (IP3) in murine peritonealmacrophages exposed to either ApoE3 (squares) or ApoE4 (circles). Thegraph shows the percent change in IP3 content in treated cells comparedto control cells exposed to vehicle but not ApoE.

FIG. 4 graphs production of TNFα (picogram/ml) by microglia primarycultures from ApoE-deficient mice after addition of peptides of SEQ IDNO:6 (squares), or addition of peptides of SEQ ID NO:6 and LPS (100ng/ml) (circles). Peptides were added in doses of 10 μM, 100 μM and 1000μM.

FIG. 5 is a graph of the optical density of cell cultures, as a measureof cell viability. Cultures of microglia from ApoE-deficient mice wereexposed to either peptides of SEQ ID NO:6 (squares), or peptides of SEQID NO:6 and LPS (100 ng/ml) (circles). Peptides were added in doses of10 μM, 100 μM and 1000 μM.

FIG. 6 graphs production of TNFα (picogram/ml) by microglia primarycultures from ApoE-deficient mice after addition of peptides of SEQ IDNO:6 (squares), or addition of peptides of SEQ ID NO:6 and LPS (100ng/ml) (circles). Peptides were added in doses of 1 μM, 10 μM, 100 μMand 1000 μM.

FIG. 7 is a graph of the optical density of cell cultures, as a measureof cell viability. Cultures of microglia from ApoE-deficient mice wereexposed to either peptides of SEQ ID NO:6 (squares), or peptides of SEQID NO:6 and LPS (100 ng/ml) (circles). Peptides were added in doses of10 μM, 100 μM and 1000 μM.

FIG. 8. Changes in [Ca²⁺]_(i) in macrophages treated with apoE. Panel A:Changes in [Ca²⁺]_(i) in a single Fura-2/AM loaded peritoneal macrophageon stimulation with apoE (100 pM). Details for measuring [Ca²⁺]_(i) aredescribed in the Examples below. The graph shown is representative of 5individual experiments using 20-30 cells each. Approximately 70-80% ofthe macrophage demonstrated changes in [Ca²⁺]_(i) upon stimulation withapoE. The arrow indicates the time of addition of apoE. Panel B: Effectof apoE concentration on changes in [Ca²⁺]_(i). The changes in[Ca²⁺]_(i) in individual cells were measured prior to and followingexposure to varying concentrations of apoE. The data are displayed asmean (S.E.) and are representative of two independent experiments; ineach case 25-30 cells were analyzed cells per study.

FIG. 9. Changes in IP₃ in macrophages treated with apoE. Panel A: Effectof apoE on IP₃ synthesis in macrophages, and modulation by pertussistoxin. These results are representative of two independent experimentsperformed in duplicate and expressed as % change in IP₃ formation atdifferent time periods in myo-[2-³H]inositol-labeled cells stimulatedwith apoE (100 pM) in the presence (open circles) and absence (filledcircles) of pertussis toxin. Panel B: Effect of apoE concentration onIP3 formation in [³H]labeled macrophages. The cells were stimulated withvarying concentrations of apoE for 60 s and IP₃ determined. Results aredisplayed as mean (S.E.) and are representative of two individualexperiments performed in duplicate.

FIG. 10A shows the performance of mice with and without treatment onrotorod latency after closed head injury.

FIG. 10B shows the weight gain of mice with and without treatment afterclosed head injury.

FIG. 10C shows the performance of mice with and without treatment in awater maze latency test after closed head injury.

FIG. 11 shows the dose-response effect of human recombinant ApoE3 onNMDA-induced cell damage. Values=mean±s.d., N=6 culture wells percondition. *=P<0.05 compared to NMDA without ApoE3. Details formeasuring NMDA-induced cell damage are provided in Examples below.

FIG. 12(A) is a schematic of full-length ApoE and ApoE-mimetic peptides.ApoE is represented by the open box. The 10-kDa lipid-binding domain islocated at the carboxyl terminus and is denoted by the shaded region.The approximate region corresponding to the ApoE LDL receptor-bindingdomain is depicted by the solid box (amino acids 130-150, SEQ ID NO:13),followed by the sequences of the three truncated apoE-mimetic peptidesused in these studies (SEQ ID NO: 10-12). (B) Circular dichroism spectraof ApoE peptides were recorded on an Aviv Model 202 CD spectrometer,using 0.1 cm pathlength cuvettes. CD spectra of the three peptides wereconsistent with a mixture of helical and random coil structure.

FIG. 13 contains graphs showing the dose response effect of ApoE peptide(133-149) (SEQ ID NO:10) on NMDA-induced cell damage of primary mixedneuronal-glial cultures at (A) 100 μM NMDA or (B) 300 μM NMDA.Values=mean±s.d., N=6-8 culture wells per condition. ═P<0.05 compared toNMDA without peptide.

FIG. 14 is a graph showing the effect of truncations of ApoE peptide onNMDA-induced cell damage. Values=mean±s.d., N=8 culture wells percondition. *=P<0.05 compared to NMDA without peptide.

FIG. 15 is a graph showing the effect of ApoE peptide (133-149) onNMDA-induced Ca⁺⁺ uptake by primary mixed neuronal-glial cultures.Values=mean±s.d., N=8 culture wells per condition. *=P<0.05 compared toNMDA without peptide.

FIG. 16 is a graph showing the effect of timing of ApoE peptide(133-149) exposure on NMDA-induced cell damage. Cultures werepre-treated (peptide added 24 h prior to and removed immediately beforeNMDA exposure), treated concurrently (peptide added immediately prior toand removed immediately after NMDA exposure), or post-treated (peptideadded immediately after NMDA exposure and maintained in the medium untildetermination of LDH released from damaged cells 24 h later) with 6 μMapoE peptide (133-149). Values=mean±s.d., N=8 culture wells percondition. *=P<0.05 compared to NMDA without peptide.

FIG. 17 contains graphs depicting the suppression of LPS-induced serumlevels of TNFα (A) and IL-6 (B) by ApoE peptide (133-149). The dark barsrepresent vehicle-treated animals and the light bars representpeptide-treated animals.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors determined that apoE modulates the activation ofglia in the CNS, and further identified several peptides that suppressesthe activation of microglia. While not wishing to be bound to a singletheory, the present inventors hypothesized that ApoE binding to amicroglial receptor affects the phenotype of the microglia, decreasingthe responsiveness of the microglia to various activators, and thereforedecreasing the release of inflammatory compounds from the microglia thatwould otherwise occur in the presence of such activators. The ApoE maybe binding to the same receptor as is bound by the activating compounds,or may be binding to a receptor independent from that bound byactivators.

In lymphocytes, ApoE has been shown to block activation by a variety ofcompounds, including LPS, the lectin PHA, and anti-CD3 antibody; theseactivators are known to bind to distinct receptors on lymphocytes. Themethods and compounds of the present invention are designed to preventor suppress the receptor-mediated activation of microglia, and thusprevent or reduce the deleterious neurological effects associated withactivated microglia. Peptides and other therapeutic molecules accordingto the present invention are able to bind to receptors on glia, anddecrease the responsiveness of the cell to various activators. In thismanner, methods and compounds according to the present invention may beused to treat, ameliorate, or prevent certain signs, symptoms, and/ordeleterious neurological effects of acute and/or chronic CNS injury.

Glutamate and related excitatory amino acids are released by synapses inthe mammalian brain, and activate ion channel glutamate receptorsincluding the NMDA, AMPA(α-amino-3-hyrdoxy-5-methyl-isoxazole-4-propionate) and kainatereceptors. Overstimulation of ionotropic glutamate receptors,particularly NMDA receptors, has been implicated in neuronaldegeneration. Systemic administration of non-competitive inhibitors suchas the NMDA receptor antagonist MK-801 prior to ischemia has been shownto prevent microglial activation, as well as delayed death of neurons,suggesting that early blockage of the glutamate cascade preventsmicroglial activation involved in ischemic injury (Streit et al., 1992).However, recent studies using a peptide (141-149) from the receptorbinding region of ApoE suggest that such peptides amplify NMDA-inducedexcitotoxicity and induce neuronal cell death (Tolar et al., 1999).Other recent studies failed to demonstrate any neuroprotective effectagainst NMDA-induced excitotoxicity from the intact apoE protein (Jordanet al., 1998; Lendon et al., 2000).

In contrast to recent reports in the literature, the present inventorshave found that intact ApoE exhibited a modest dose-dependent reductionin NMDA induced cytotoxicity. By comparison, a seventeen residueApoE-mimetic peptide (SEQ ID NO:10) surprisingly exhibited asignificantly more robust neuroprotection relative to native ApoE andblocked both the calcium influx and cell death associated with NMDAexposure as completely as the NMDA receptor antagonist MK-801. Furthertruncation of the peptide at the amino terminal resulted in aprogressive loss of neuroprotection from NMDA excitotoxicity. Theseresults suggest that one way which ApoE affects recovery of neuronalcells from ischemic injury following brain insult is by protecting cellsagainst glutamate toxicity. Furthermore, the results support the use ofApoE-mimetic peptides as a valuable therapeutic strategy followingcerebral ischemia.

The unexpected finding that ApoE peptides completely suppress theneuronal cell death and calcium influx associated withN-methyl-D-aspartate exposure provides the basis for ApoE-basedformulations and treatments for disorders and diseases associated withactivation of glutamate receptors which would not have been apparentbefore the present invention. The finding also provides the basis forcombined therapeutic compositions containing one or more more of thepeptides or NMDA antagonist compounds of the invention in combinationwith known reagents for treating diseases associated with NMDAexcitotoxicity.

For instance, NMDA excitotoxicity has been associated with HIV dementiaand encephalopy (Perez et al., 2001; Haughey et al., 2001; Doble, 1999).The fact that ApoE peptides work as NMDA antagonists is particularlysurprising seeing as no statistically significant correlation has beenfound between the risk of HIV dementia or HIV encephalitis in relationto apoE genotypes (Dunlop et al., 1997). Thus, even without the recentreports that ApoE enhances NMDA show NMDA antagonistic activity.

NMDA excitotoxicity has also been associated with neurolathyrism,amyotrophic lateral sclerosis (ALS) (Doble, 1999; Nguimfack, 2002),schizophrenia, Huntington's chorea, Parkinson's (Nguimfack, 2002;Mytilineou et al., 1997; Klopman and Sedykh, 2002; Le and Lipton, 2001),bipolar disorder (Farber et al. 2002), multiple sclerosis in humans andexperimental allergic encephalomyelitis (EAE) in animals (Paul andBolton, 2002), depression, stroke (Le and Lipton, 2001), epilepsy andthe inherited neurometabolic disease d-2-hydroxyglutaric aciduria(Kolker et al., 2002), in addition to Alzheimer's Disease (Bi et al.,2002; Bi and Sze, 2002) and traumatic brain injury (Rao et al., 2001;Regner et al., 2001; Xu and Luo, 2001). NMDA antagonists are also usedin clinical anesthesia (Farber et al., 2002), and have been shown toinhibit chronic pain (McKenna and Melzack, 2001; Le and Lipton, 2001),drug tolerance (Cady, 2001) and alcohol dependency in an animal model(Kotlinska, 2001).

Thus, the present invention includes the use of the disclosed peptidesand NMDA antagonist compounds in methods and pharmaceutical formulationsfor the treatment of any of the above diseases or disorders, and asingredients in anesthesia formulations and in combined therapeuticcompositions containing other known compounds useful for treating thevarious disorders. For instance, the peptides and other compounds of theinvention may be combined with any known HIV drug, including HIV reversetranscriptase and protease inhibitors, in a combined therapeutic regimengeared toward inhibiting viral replication and preventing or treatingHIV dementia, or may be administered alone or with other NMDAantagonists in a supplementary formulation. One author recentlycommented that, even though antiretroviral therapy of the CNS isessential for improvement in function and prognosis in patientsdemonstrating AIDS dementia complex, it may also be necessary in thelong term to provide additional neuroprotection, blocking secondarymechanisms of neurotoxicity, since a significant portion of toxicityseems to be mediated by indirect mechanisms that continue even duringantiretroviral therapy (Clifford, 2002).

Riluzole is a substance with glutamate antagonistic properties that isused for neuroprotective treatment in amyotrophic lateral sclerosis andwhich is currently being tested in clinical trials for treatment ofHuntington's disease and Parkinson's disease (Schiefer et al., 2002;Doble, 1999). Schiefer and colleagues recently demonstrated thatRiluzole prolongs survival time and alters nuclear inclusion formationin a transgenic mouse model of Huntington's disease. Thus, given theNMDA antagonistic role of the peptides and compounds of the invention,these peptides and compounds could be used in pharmaceuticalformulations for the treatment of ALS, Huntington's and Parkinson's,alone or in combination with other glutamate antagonists such asRiluzole.

L-deprenyl is an inhibitor of monoamine oxidase (MAO)-B that delays theemergence of disability and the progression of signs and symptoms ofParkinson's disease, and is predicted to exert a protective effect fromevents occurring downstream from activation of glutamate receptors(Mytilineou et al., 1997). MAO-B inhibitors, dopamine receptorantagonists such as Levodopa and NMDA receptor antagonists have all beenshown to have an antiparkinson effect, and multidrug combinations havebeen shown to synergistically enhance the antiparkinson effects of thedrugs (Klopman and Sedykh, 2002). Thus, given the NMDA antagonistic roleof the peptides and compounds of the invention, these peptides andcompounds could be used in pharmaceutical formulations for the treatmentof Parkinson's, alone or in combination with other NMDA receptorantagonists, MAO-B inhibitors such as L-deprenyl and dopamine receptorantagonists such as Levodopera.

The production of free radicals as a result of glutamate excitotoxicityhas been implicated in the pathogenesis of schizophrenia (Nguimfack,2002). Thus, researchers have begun to examine treatment ofschizophrenia with antioxidizing substances used in other neurologicaldiseases such as ALS, Parkinson's and Huntington's disease. Given thatthe NMDA receptor antagonistic peptides and compounds of the inventionmay be used to inhibit the production of free radicals as a result ofglutamate excitotoxicity, these peptides and compounds may be used inpharmaceutical formulations for the treatment of schizophrenia, alone orin combination with other antioxidizing substances.

Anticonvulsant, antiepileptic agents that inhibit NMDA receptorhypofunction have found to be of clinical use in bipolar disorder(Farber et al., 2002). Such agents include phenyloin, carbamazepine,valproic acid, lamotrigine, riluzole, tetrodotoxin, felbamate,gabapentin and ethosuximide. Given that the peptides of the compounds ofthe present invention also inhibit NMDA receptor-associatedneurotoxicity, the peptides and compounds of the present invention maybe used alone or in combination with other NMDA receptor antagonists orinhibitors of NMDA receptor hypofunction in pharmaceuticals and methodsof treating bipolar disorder or epilepsy.

Multiple sclerosis (MS) is an immunologically mediated disease, asdetermined by observation of the response to immunotherapy and theexistence of an animal model, experimental autoimmune encephalitis.Interferon (IFN) beta-1b, IFN beta-1a, and glatiramer acetate, currenttherapies used for relapsing or remitting MS, have mechanisms of actionthat address the immunologic pathophysiology of MS (Dhib-Jalbut, 2002).For instance, the IFNs bind to cell surface-specific receptors,initiating a cascade of signaling pathways that end with the secretionof antiviral, antiproliferative, and immunomodulatory gene products.Glatiramer acetate, a synthetic molecule, inhibits the activation ofmyelin basic protein-reactive T cells and induces a T-cell repertoirecharacterized by anti-inflammatory effects. Several currently marketedtreatments, including IV immunoglobulin, methotrexate, and azathioprine,are being evaluated as treatments for relapsing-remitting multiplesclerosis (RRMS) in combination with the approved therapies (Calabrese,2002). Given that the NMDA receptor antagonist memantine has been shownto prevent and breakdown of and restore the blood-brain barrier andreduce symptoms associated with pathogenesis of EAE in vivo (Paul andBolton, 2002), the peptides and compounds of the present invention maybe used alone or in combination with other NMDA receptor antagonists orin addition to interferons or Glatiramer acetate for the treatment of MSin humans.

Using an animal model of persistent human pain, McKenna and Melzackrecently showed that pain behavior was significantly reduced bytreatment with the NMDA receptor antagonist AP5 (McKenna and Melzack,2001). Similarly, Von Bergen and colleagues recently demonstrated thatintrathecal administration of LY293558, a competitivenon-N-methyl-D-aspartate excitatory amino acid receptor antagonist,blocked both sensory and motor responses in rats through 180 min, withcomplete recovery observed the following day. The effects of LY293558were more pronounced and sustained than those of bupivacaine, leadingthe authors to conclude that drugs like LY293558 that block glutamatereceptors may be an alternative to local anesthetics for spinalanesthesia in humans (Von Bergen et al., 2002). Thus, the peptides andcompounds of the present invention may be used alone or in combinationwith other NMDA receptor antagonists or in addition to other anestheticcompounds as local anesthetics in humans and animals.

NMDA receptors are also believed to play a major role in thepathophysiology of substance use (Kotlinska, 2001; Soyka et al., 2000).For instance, Kotlinska showed that the NMDA receptor antagonistmemantine given prior to ethanol administration prevented thedevelopment of ethanol dependence in rats. Jones and colleaguesdemonstrated that the intensity of morphine withdrawal syndrome wasreduced in rat pups pre-treated with the NMDA receptor antagonist,LY235959. Withdrawal behaviors such as head moves, moving paws, rolling,and walking were decreased, and vocalizations were completely eliminatedin pups pre-treated with LY2359559 (Jones et al., 2002). According to arecent review, strategies aimed at targeting the basic mechanisms ofaddiction rely on the premise that addiction is caused by adaptivechanges in the central nervous system and that craving, which is themain cause of relapse, depends on dopaminergic mechanisms and requireshigh general excitability. Thus, pharmacological approaches haveinvolved drugs that reduce neuronal adaptability by inhibiting thecalcium entry to neurons both through voltage-gated channels (e.g.nimodipine) and NMDA receptors (e.g. memantine), as well as drugs thatstimulate the inhibitory GABAergic system (gamma-vinyl-GABA, baclofen).Thus, the peptides and compounds of the present invention may be usedalone or in combination with other NMDA receptor antagonists such asmemantine or in addition to other neuronal adaptability compounds suchas nimodipine, gamma-vinyl-GABA and baclofen in compositions and methodsfor the prevention and treatment of alcohol and drug addiction inhumans.

The finding by the present inventors that one way in which ApoE peptidesinhibit microglial activation is by inhibiting glutamate excitotoxicitylends further support to the value of these peptides and the other NMDAreceptor antagonistic compounds of the invention for the treatment ofCNS injury. For instance, Rao et al. reported neuroprotection bymemantine, another NMDA receptor antagonist, after traumatic braininjury in rats (Rao et al., 2001). Other authors recently commented thatexcessive activation of NMDA receptors may be one of the most importantfactors to induce secondary cerebral impairments, and NMDA receptorantagonists such as AP5 may protect the brain from edema after braininjury. Thus, the peptides and compounds of the present invention may beused alone or in combination with other NMDA receptor antagonists incompositions and methods for the treatment of brain injury andassociated secondary cerebral impairments in humans and animals.

The present methods and compounds are useful in preventing, treating, orameliorating neurological signs and symptoms associated with acute CNSinjury. As used herein, acute CNS injury includes but is not limited tostroke (caused by thrombosis, embolism or vasoconstriction), closed headinjury, global cerebral ischemia (e.g., ischemia due to systemichypotension of any cause, including cardiac infarction, cardiacarrhythmia, hemorrhagic shock, and post coronary artery bypass graftbrain injury), focal ischemia and intracranial hemorrhage. Ischemicdamage to the central nervous system may result from either global orfocal ischemic conditions. Global ischemia occurs where blood flow tothe entire brain ceases for a period of time, such as during cardiacarrest. Focal ischemia occurs when a portion of the brain is deprived ofnormal blood flow, such as during thromboembolytic occlusion of acerebral vessel, traumatic head injury, edema and brain tumors. Much ofthe CNS damage due to cerebral ischemia occurs during the hours or evendays following the ischemic condition, and is secondary to the releaseof cytotoxic products by damaged tissue.

The present methods and compounds are also useful in preventing,treating, or ameliorating neurological signs and symptoms associatedwith chronic neurological disease, including but not limited toAlzheimer's disease (AD) and HIV-associated encephalopathy. The findingby the present inventors that ApoE peptides may be used to suppressglial activation provides a role for the peptides and compounds of theinvention in the treatment of any neurological disease involvingmicroglial activation. For example, microglia express markers ofactivation in AD, suggesting that crucial inflammatory events in ADinvolve microglia. Such activated microglia cluster near amyloid plaques(Griffin et al., 1995). Microglia are also activated in epilepsy (Shenget al., 1994).

The surprising finding by the present inventors that one way in whichApoE peptides inhibit peptides and other compounds of the invention fortreating AD, since it has been recently shown that uptake and pathogeniceffects of amyloid beta peptide are blocked by NMDA receptor antagonists(Bi et al., 2002). Other studies indicate that anti-inflammatory drugsmay delay the onset or progression of the disease (Breitner et al.,1995; Rogers et al., 1993). Thus, the peptides and compounds of thepresent invention may be used alone or in combination with other NMDAreceptor antagonists or other known pharmaceuticals and especiallyanti-inflammatory drugs used for the treatment of AD in compositions andmethods for the treatment of AD in humans.

The present methods and compounds are also useful in preventing,treating, or ameliorating the neurological signs and symptoms associatedwith inflammatory conditions affecting the nervous system including theCNS, including but not limited to multiple sclerosis, vasculitis, acutedisseminated encephalomyelitis, and Guillain-Barre syndrome. In thisregard, the ApoE peptides and other compounds of the invention may beused alone or in combination with other known anti-inflammatory drugs orcytokines to formulate pharmaceutical compositions for the treatment ofCNS inflammatory conditions.

The present methods and compounds are useful in preventing, suppressingor reducing the activation of glia in the CNS that occurs as a part ofacute or chronic CNS disease. The effect of the present methods andcompounds may be assessed at the cellular or tissue level (e.g.,histologically or morphometrically), or by assessing a subject'sneurological status. The suppression or reduction of glial activationcan be assessed by various methods as would be apparent to those in theart; one such method is to measure the production or presence ofcompounds that are known to be produced by activated glia, and comparesuch measurements to levels of the same compounds in control situations.Alternatively, the effects of the present methods and compounds insuppressing, reducing or preventing microglial activation may beassessed by comparing the signs and/or symptoms of CNS disease intreated and control subjects, where such signs and/or symptoms areassociated with or secondary to activation of microglia.

The present invention is also based on the surprising finding by theinventors that ApoE receptor binding peptides protect againstLPS-induced production of cytokines in the periphery in an in vivoanimal model of sepsis. Although intact ApoE has recently been shown toprotect mice from bacterial LPS-induced lethality (Van Oosten et al.,2001), it is surprising that a peptide containing only the receptorbinding region of ApoE confers protection given that ApoE is thought tomediate protection by redirecting LPS from macrophages to parenchymalliver cells. Other possible therapies for sepsis involve theadministration of anti-inflammatory beta, granulocyte colony-stimulatingfactor, IFN-phi, macrophage migration inhibitory factor and highmobility group 1 protein (Zanotti et al., 2002), and monoclonalantibodies, including antiendotoxin antibodies, anti-tumor necrosisfactor antibodies, and anti-CD14 antibodies (Matsubara et al., 2002).Thus, the peptides and compounds of the present invention may be usedalone or in combination with other known anti-inflammatory cytokines andantibodies in compositions and methods for the treatment of sepsis.

As used herein, the terms “combating,” “treating” and “ameliorating” arenot necessarily meant to indicate a reversal or cessation of the diseaseprocess underlying the CNS or sepsis condition afflicting the subjectbeing treated. Such terms indicate that the deleterious signs and/orsymptoms associated with the condition being treated are lessened orreduced, or the rate of progression is reduced, compared to that whichwould occur in the absence of treatment. A change in a disease sign orsymptom may be assessed at the level of the subject (e.g., the functionor condition of the subject is assessed), or at a tissue or cellularlevel (e.g., the production of markers of glial or macrophage activationis lessened or reduced). Where the methods of the present invention areused to treat chronic CNS conditions (such as Alzheimer's disease), themethods may slow or delay the onset of symptoms such as dementia, whilenot necessarily affecting or reversing the underlying disease process.

Suitable subjects for carrying out the methods of the present inventioninclude male and female mammalian subjects, including humans, non-humanprimates, and non-primate mammals. Subjects include veterinary(companion animal) subjects, as well as livestock and exotic species.

Active compounds that may be used in the methods of the presentinvention include ligands or agonists that specifically and/orselectively bind to the LRP/α2M receptor or to any receptor bound by theApoE peptides of the invention. Examples of such compounds include, butare not limited to, 1) alpha 2 macroglobulin; 2) pseudomonas exotoxin;3) lipoprotein lipase; 4) apolipoprotein E; 5) oxidized and/oracetylated LDL; 6) receptor associated protein (RAP); 7) remnantparticles; 8) low density lipoprotein (LDL); 9) high density lipoprotein(HDL); 10) lactoferrin; 11) tissue plasminogen activator (tPA); 12)urine plasminogen activator (uPA); etc., and receptor binding fragmentsthereof.

As used herein, an “ApoE peptide” or a “peptide of ApoE” refers to anypeptide of ApoE or functional variant thereof that binds to a receptorbound by ApoE and mediates the functional effects described herein.Amino acid residues 100-200 of each isoform of the ApoE moleculecomprise a known ApoE receptor binding region. More specifically, thereceptor binding region of ApoE is within amino acid residues 130-160 ofeach isoform of the ApoE molecule (SEQ ID NO:4 and SEQ ID NO:5), andmore specifically is within amino acid residues 140-155 (HLRKLRKRLLRDADDL) (SEQ ID NO:1). See, e.g., Weisgraber, Apolipoprotein E:Structure-Function Relationships, Advances in Protein Chemistry 45:249(1994). The amino acid interchanges that define the E2, E3 and E4isoforms are not found within the region of amino acid residues 140-155,but do influence the overall structure of the Apolipoprotein molecule.ApoE2 and ApoE3 molecules form covalently bound homodimers; ApoE4molecules do not.

As used herein, the term homodimer refers to a molecule composed of twomolecules of the same chemical composition; the term heterodimer refersto a molecule composed of two molecules of differing chemicalcomposition.

The present inventors utilized a 9-mer monomer having an amino acidsequence LRKLRKRLL (SEQ ID NO:2). This 9 amino acid sequence is foundwithin the larger ApoE receptor binding sequence region identifiedabove, and is found at amino acid positions 141-149 of ApoE. The presentinventors constructed a dimer of SEQ ID NO:2, i.e., a peptide having anamino acid sequence of LRKLRKRLL LRKLRKRLL (SEQ ID NO:3). Peptides ofSEQ ID NO:3 suppressed microglial activation in a dose-dependentfashion. Use of the monomer (monomer peptides of SEQ ID NO:2) did notsuppress microglial activation. (See FIG. 2).

The present inventors further utilized a 20-mer monomer having an aminoacid sequence TEELRVRLAS HLRKLRKRLL (SEQ ID NO:6). This 20 amino acidsequence is found at amino acid positions 130-149 of ApoE, and comprisesthe 9-mer SEQ ID NO:2. Peptides of SEQ ID NO:6 suppressed microglialactivation in a dose-dependent fashion (see FIGS. 4-7).

The present inventors further showed that a 17-mer having the amino acidsequence LRVRLAS HLRKLRKRLL (SEQ ID NO:10) from amino acid positions133-149 of ApoE was protective in a murine head injury model and in amurine model of LPS-induced sepsis. The same peptide was also shown toinhibit NMDA excitotoxicity in primary rat neuronal/glial cell cultures.

In contrast, Clay et al., Biochemistry 34:11142 (1995) reported thatdimeric peptides of amino acids 141-155 or 141-149 were both cytostaticand cytotoxic to T lymphocytes in culture. Cardin et al. Biochem BiophysRes. Commun. 154:741 (1988) reported that a peptide of apoE 141-155inhibited the proliferation of lymphocytes. A peptide consisting of atandem repeat of amino acids 141-155, as well as longer monomericpeptides comprising the 141-155 region, was found to cause extensive andspecific degeneration of neurites from embryonic chicks in vitro.Crutcher et al., Exp. Neurol. 130:120 (1994). These authors suggestedthat peptide sequences associated with apoE might contribute directly toneurodegenerative processes, thereby supporting the unexpected nature ofthe neuroprotective effect achieved with the peptides of the presentinvention.

Peptides of the present invention may be produced by standard techniquesas are known in the art. Peptides useful in the present methods includethose comprising the ApoE LDL receptor binding sequence (includingmultiple repeats thereof, including but not limited to dimers andtrimers); and conjugates of two or more peptides, each of whichcomprises a peptide as described herein or a peptide comprising the LDLreceptor binding sequence. One ApoE receptor binding sequence isprovided in SEQ ID NO:1. A preferred peptide comprises or consists ofmultiple repeats of SEQ ID NO: 2, preferably dimers thereof. Thus, apreferred peptide useful in the present methods is SEQ ID NO:3 (a tandemrepeat of LRKLRKRLL), or peptides comprising SEQ ID NO:3. Furtherpreferred peptides comprise or consist of SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6 or SEQ ID NO:10.

Modification of the peptides disclosed herein to enhance the functionalactivities associated with these peptides could be readily accomplishedby those of skill in the art. For instance, the ability of a lineartandem repeat of amino acids 141-155 (the 141-155 dimer) to bind the LDLreceptor was studied by Dyer et al., J. Lipid Research 36:80 (1995). Aseries of modified peptides was constructed and assessed for LDL bindingability. These authors report that deletion of the charged aminoterminal residues (including arg142 and lys143) in 145-155 or 144-150dimers abolished the LDL receptor activities of the peptides. Theseauthors conclude that LDL-receptor binding activity of the 141-155 dimeris dependent on at least two clusters of basic amino acids present onthe hydrophilic face of the amphipathic alpha-helix of the 141-155,141-150, 141-155 (lys143→ala) and 141-155 (arg150→gala) dimer peptides.Dyer et al., J. Biol. Chem. 266:15009 (1991) reported that aself-conjugate of peptide 141-155, and a peptide consisting of a tandemrepeat of 141-155, were able to inhibit both lymphocyte proliferationand ovarian androgen production. Dyer et al., J. Biol. Chem. 266:22803(1991) investigated the LDL binding ability of a dimeric 141-155 tandempeptide, and a trimeric 141-155 peptide. Binding was decreased withamino acid substitutions of Lys-143-→Ala, Leu144-→Pro, and Arg150-→Ala.Lalazar et al., J. Biol. Chem. 263:3542 (1988) investigated variants ofApoE for binding to the LDL receptor. When neutral amino acids weresubstituted for basic residues at positions 136, 140, 143, and 150,binding activity was reduced. Where proline was substituted forleucine144 or alanine152, binding was reduced. However, slightlyenhanced receptor binding was displayed by a variant in which argininewas substituted for serine139 and alanine was substituted for leucine149.

Active compounds (or “active agents”) useful in the methods of thepresent invention include those that compete with a peptide of SEQ IDNO:3, and/or a peptide of SEQ ID NO:6, and/or a peptide of SEQ ID NO:10in binding to microglial receptors or receptors on neighboring effectorcells, such as astrocytes, to thereby prevent or suppress activation ofthe microglia by molecules that would otherwise activate microglia.Compounds that are useful in the present methods also include thosewhich act as antagonists for the receptor bound by peptides of SEQ IDNO:3 and/or SEQ ID NO:6 and/or SEQ ID NO:10. Antibodies that selectivelytarget and bind to this receptor can also be used as antagonists ofmicroglial activation according to the present invention. Suchantibodies selectively or specifically bind to the receptor bound bypeptides of SEQ ID NO:3 and/or peptides of SEQ ID NO:6 and/or peptidesof SEQ ID NO:10.

Peptides of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:10 or conformationalanalogues thereof, are an aspect of the present invention. Suchcompounds are peptides or peptidomimetics having a core sequence ofamino acids with a conformation in aqueous solution that interacts withreceptor molecules on glial cells to block the activation of glial cellsthat would otherwise occur in conjunction with acute or chronic CNSinjury, or exposure to known activators of microglia such as LPS. Statedanother way, such compounds are characterized by the ability to competewith peptides of SEQ ID NO:3 and/or peptides of SEQ ID NO:6 and/or SEQID NO:10 for binding to microglia, and by their ability to suppressmicroglial activation by known activators such as LPS.

Another variation of the therapeutic peptides of the present inventionis the linking of from one to five amino acids or analogues to theN-terminal or C-terminal amino acid of the therapeutic peptide. Analogsof the peptides of the present invention may also be prepared by addingfrom one to five additional amino acids to the N-terminal, C-terminal,or both N- and C-terminals, of an active peptide, where such amino acidadditions do not adversely affect the ability of the peptide to bind tomicroglia at the site bound by a peptide of SEQ ID NO:3 and/or SEQ IDNO:6 and/or SEQ ID NO:10.

Changes in the amino acid sequence of peptides can be guided by knownsimilarities among amino acids and other molecules or substituents inphysical features such as charge density, hydrophobicity,hydrophilicity, size and configuration, etc. For example, the amino acidThr may be replaced by Ser and vice versa, and Leu may be replaced byIle and vice versa. Further, the selection of analogs may be made bymass screening techniques known to those skilled in the art (e.g.,screening for compounds which bind to microglia at the receptor bound bya peptide of SEQ ID NO:3 and/or SEQ ID NO:6 and/or SEQ ID NO:10). Apreferred exchange is to replace Ser with Arg, to increase the argininecontent of the peptide; examples include peptides of or comprising SEQID NO:7, SEQ ID NO:8 or SEQ ID NO:9. A further preferred exchange is tosubstitute alanine for leucine149.

Peptides of the present invention may also be characterized as shortpeptides of from about 20 amino acids, 22 amino acids, 24 amino acids,26 amino acids, 28 amino acids, 30 amino acids, 35 amino acids, or 40amino acids, up to about 22 amino acids, 24 amino acids, 26 amino acids,28 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 aminoacids, 50 amino acids or more, where the peptides comprise the 18-aminoacid sequence LRKLRKRLL LRKLRKRLL (SEQ ID NO:3), or variants thereofthat retain the receptor binding ability of peptides of SEQ ID NO:3. Apreferred peptide useful in the present invention is one consisting ofor comprising SEQ ID NO:3. Where longer peptides are employed, thoseincorporating amino acid sequences derived from the ApoE sequenceimmediately surrounding amino acid residues 141-149 are preferred. Wherepeptides longer than 18 amino acids are employed, it is contemplatedthat they may include virtually any other amino acid sequences so longas the resultant peptide maintains its ability to bind to microglial andsuppress microglia activation in acute and chronic CNS inflammation. Thepresent invention includes those variations of the ApoE sequence at141-149 which are known to retain the ability LDL receptor-bindingability. Synthetic peptides may further be employed, for example, usingone or more D-amino acids in place of L-amino acids, or by adding groupsto the N- or C-termini, such as by acylation or amination.

Peptides of the present invention may also be characterized as shortpeptides of from about 10 amino acids, 12 amino acids, 14 amino acids,15 amino acids, 18 amino acids, 20 amino acids, 22 amino acids, 24 aminoacids, 26 amino acids, 28 amino acids, 30 amino acids, 35 amino acids,or 40 amino acids, up to about 15 amino acids, 22 amino acids, 24 aminoacids, 26 amino acids, 28 amino acids, 30 amino acids, 35 amino acids,40 amino acids, 45 amino acids, 50 amino acids or more, where thepeptides comprise the 9-amino acid sequence LRKLRKRLL (SEQ ID NO:2), orvariants thereof that retain the receptor binding ability of peptides ofSEQ ID NO:3 and/or SEQ ID NO:6 and/or SEQ ID NO:10. A preferred peptideuseful in the present invention is one consisting of or comprising theapoE receptor binding region; a particularly preferred peptide consistsof or comprises SEQ ID NO:6 and/or SEQ ID NO:10. Where longer peptidesare employed, those incorporating amino acid sequences derived fromwithin the apoE receptor binding region, or the ApoE sequenceimmediately surrounding the apoE receptor binding region, are preferred,although it is contemplated that these peptides may include virtuallyany other amino acid sequences so long as the resultant peptidemaintains its ability to bind to microglia and suppress microgliaactivation in acute and chronic CNS inflammation. The present inventionincludes those variations of the ApoE sequence at 141-149 which areknown to retain the ability LDL receptor-binding ability. Syntheticpeptides may further be employed, for example, using one or more D-aminoacids in place of L-amino acids, or by adding groups to the N- orC-termini, such as by acylation or amination.

The peptides of the present invention include not only natural aminoacid sequences, but also peptides which are analogs, chemicalderivatives, or salts thereof. The term “analog” or “conservativevariation” refers to any polypeptide having a substantially identicalamino acid sequence to the therapeutic peptides identified herein, andin which one or more amino acids have been substituted with chemicallysimilar amino acids. For example, a polar amino acid such as glycine orserine may be substituted for another polar amino acid; a basic aminoacid may be substituted for another basic amino acid, or an acidic aminoacid may be substituted for another acidic amino acid; or a non-polaramino acid may be substituted for another non-polar amino acid. Thereterm “analog” or “conservative variation” as used herein also refers toa peptide which has had one or more amino acids deleted or added to apolypeptide of the present invention, but which retains a substantialsequence similarity (at least about 85% sequence similarity, andpreferably at least 90%, 92%, 94%, 95%, 96%, 98% or even 99% sequencesimilarity), where the peptide retains the ability to suppressmicroglial activation as described herein.

The amino acids constituting peptides of the present invention may be ofeither the L-configuration or the D-configuration. Therapeutic peptidesof the present invention may be in free form or the form of a salt,where the salt is pharmaceutically acceptable.

As used herein, the term “administering to the brain of a subject”refers to the use of routes of administration, as are known in the art,that provide the compound to the central nervous system tissues, and inparticular the brain, of a subject being treated.

Preferably, the compounds of the present invention are used incombination with a pharmaceutically acceptable carrier. The presentinvention thus also provides pharmaceutical compositions suitable foradministration to mammalian subjects. Such compositions comprise aneffective amount of the compound of the present invention in combinationwith a pharmaceutically acceptable carrier. The carrier may be a liquid,so that the composition is adapted for parenteral administration, or maybe solid, i.e., a tablet or pill formulated for oral administration.Further, the carrier may be in the form of a nebulizable liquid or solidso that the composition is adapted for inhalation. When administeredparenterally, the composition should by pyrogen free and in anacceptable parenteral carrier. Active compounds may alternatively beformulated encapsulated in liposomes, using known methods. Additionally,the intranasal administration of peptides to treat CNS conditions isknown in the art (see, e.g., U.S. Pat. No. 5,567,682 to Pert, regardingintranasal administration of peptide T to treat AD). (All patentsreferenced herein are intended to be incorporated by reference herein intheir entirety.) Preparation of a Compound of the Present Invention forIntranasal Administration May be Carried out using techniques as areknown in the art.

Pharmaceutical preparations of the compounds of the present inventionmay optionally include a pharmaceutically acceptable diluent orexcipient. For the sepsis-related embodiments of the invention, thedisclosed peptides may be conjugated to pharmaceutically acceptablecarriers to increase serum half-life using methods that are known tothose of skill in the art. See, e.g., U.S. Pat. No. 6,423,685, which isherein incorporated by reference in its entirety.

An effective amount of the compound of the present invention is thatamount that decreases microglial activation compared to that which wouldoccur in the absence of the compound; in other words, an amount thatdecreases the production of neurotoxic compounds by the microglia,compared to that which would occur in the absence of the compound. Theeffective amount (and the manner of administration) will be determinedon an individual basis and will be based on the specific therapeuticmolecule being used and a consideration of the subject (size, age,general health), the condition being treated (AD, acute head injury,cerebral inflammation, etc.), the severity of the symptoms to betreated, the result sought, the specific carrier or pharmaceuticalformulation being used, the route of administration, and other factorsas would be apparent to those skilled in the art. The effective amountcan be determined by one of ordinary skill in the art using techniquesas are known in the art. Therapeutically effective amounts of thecompounds described herein may be determined using in vitro tests,animal models or other dose-response studies, as are known in the art.

The compounds of the present invention may be administered acutely(i.e., during the onset or shortly after events leading to cerebralinflammation or ischemia), or may be administered prophylactically(e.g., before scheduled surgery, or before the appearance of neurologicsigns or symptoms), or administered during the course of a degenerativedisease to reduce or ameliorate the progression of symptoms that wouldotherwise occur. The timing and interval of administration is variedaccording to the subject's symptoms, and may be administered at aninterval of several hours to several days, over a time course of hours,days, weeks or longer, as would be determined by one skilled in the art.

The typical daily regime may be from about 0.01 μg/kg body weight perday, from about 10 μg/kg body weight per day, from about 100 μg/kg bodyweight per day, from about 1000 μg/kg body weight per day, from about10,000 μg/kg body weight per day, from about 100,000 μg/kg body weightper day.

The blood-brain barrier presents a barrier to the passive diffusion ofsubstances from the bloodstream into various regions of the CNS.However, active transport of certain agents is known to occur in eitherdirection across the blood-brain barrier. Substances that may havelimited access to the brain from the bloodstream can be injecteddirectly into the cerebrospinal fluid. Cerebral ischemia andinflammation are also known to modify the blood-brain barrier and resultin increased access to substances in the bloodstream.

Administration of a compound directly to the brain is known in the art.Intrathecal injection administers agents directly to the brainventricles and the spinal fluid. Surgically-implantable infusion pumpsare available to provide sustained administration of agents directlyinto the spinal fluid. Lumbar puncture with injection of apharmaceutical compound into the cerebrospinal fluid (“spinalinjection”) is known in the art, and is suited for administration of thepresent compounds.

Pharmacologic-based procedures are also known in the art forcircumventing the blood brain barrier, including the conversion ofhydrophilic compounds into lipid-soluble drugs. The active agent may beencapsulated in a lipid vesicle or liposome.

The intra-arterial infusion of hypertonic substances to transiently openthe blood-brain barrier and allow passage of hydrophilic drugs into thebrain is also known in the art. U.S. Pat. No. 5,686,416 to Kozarich etal. discloses the co-administration of receptor mediated permeabilizer(RMP) peptides with compounds to be delivered to the interstitial fluidcompartment of the brain, to cause an increase in the permeability ofthe blood-brain barrier and effect increased delivery of the compoundsto the brain. Intravenous or intraperitoneal administration may also beused to administer the compounds of the present invention.

One method of transporting an active agent across the blood-brainbarrier is to couple or conjugate the active agent to a second molecule(a “carrier”), which is a peptide or non-proteinaceous moiety selectedfor its ability to penetrate the blood-brain barrier and transport theactive agent across the blood-brain barrier. Examples of suitablecarriers include pyridinium, fatty acids, inositol, cholesterol, andglucose derivatives. The carrier may be a compound which enters thebrain through a specific transport system in brain endothelial cells.Chimeric peptides adapted for delivering neuropharmaceutical agents intothe brain by receptor-mediated transcytosis through the blood-brainbarrier are disclosed in U.S. Pat. No. 4,902,505 to Pardridge et al.These chimeric peptides comprise a pharmaceutical agent conjugated witha transportable peptide capable of crossing the blood-brain barrier bytranscytosis. Specific transportable peptides disclosed by Pardridge etal. include histone, insulin, transferrin, and others. Conjugates of acompound with a carrier molecule, to cross the blood-brain barrier, arealso disclosed in U.S. Pat. No. 5,604,198 to Poduslo et al. Specificcarrier molecules disclosed include hemoglobin, lysozyme, cytochrome c,ceruloplasmin, calmodulin, ubiquitin and substance P. See also U.S. Pat.No. 5,017,566 to Bodor.

An alternative method of administering peptides of the present inventionis carried out by administering to the subject a vector carrying anucleic acid sequence encoding the peptide, where the vector is capableof entering brain cells so that the peptide is expressed and secreted,and is thus available to microglial cells. Suitable vectors aretypically viral vectors, including DNA viruses, RNA viruses, andretroviruses. Techniques for utilizing vector deliver systems andcarrying out gene therapy are known in the art. Herpesvirus vectors area particular type of vector that may be employed in administeringcompounds of the present invention.

Screening Methods. Also disclosed herein are methods of screeningcompounds for the ability to prevent or reduce microglial activationunder conditions of cerebral ischemia or cerebral inflammation. Suchmethods comprise contacting an activated microglial cell with a testcompound, and detecting whether the test compound binds to microglia atthe same receptor at which peptides of SEQ ID NO:3 and/or SEQ ID NO:6and/or SEQ ID NO:10 bind. The contacting step may be carried out invitro, for example in cell culture. A competitive binding assay may beused to detect whether the test compound binds to the same receptor thatis bound by peptides of SEQ ID NO:3 and/or SEQ ID NO:6 and/or SEQ IDNO:10, for instance by detecting the inhibition of receptor binding of apeptide of the invention that is conjugated to or associated with adetectable label such a radioisotope or a fluorescent molecule, or anyother detectable label that is known and commonly used in the art.

An additional method of screening a test compound for the ability tosuppress microglial activation comprises incubating an activatedmicroglial cell culture with a test compound, and measuring at least onemarker of microglial activation. A decrease in a marker of microglialactivation (compared to the level of that marker that would occur in theabsence of the test compound) indicates that the test compound is ableto suppress, prevent or reduce microglial activation. An exemplarymarker of microglial activation is the production of nitric oxide.

A further method of screening a test compound for the ability tosuppress microglial activation involves pre-incubating a microglial cellculture with a test compound, then incubating the microglial cellculture with a compound that is known to activate microglia. At leastone marker of microglial activation is then measured, and a decrease inthe activation marker (compared to that which occurs in the absence ofthe pre-incubation step) indicates that the test compound is able toaffect microglial activation. An exemplary marker of microglialactivation is the production of nitric oxide.

Atherosclerosis. It known that the inflammatory process mediates anaspect of the atherosclerotic process. See, e.g., Hansson (1994);Berliner et al. (1995); Watanabe et al. (1997). ApoE is known to besecreted by macrophages locally at blood vessel walls (although theamount secreted by macrophages in an individual is trivial compared tothe amount of ApoE produced by the liver). In the classic model ofatherosclerosis, ApoE functions to remove cholesterol from the bloodstream and deliver it to macrophages or to the liver. However, it hasbecome apparent that ApoE secreted by macrophages at the blood vesselwall decreases atherosclerotic plaque formation, independent of anylipid metabolism effects. For instance, ApoE-deficient mice are acceptedas a model of hypercholesteremia and atherosclerotic disease. ProvidingApoE-secreting macrophages to such mice dramatically decreasesatherosclerotic plaque formation. Linton et al. (1995). Conversely,replacing a wild-type mouse's macrophages with ApoE-deficientmacrophages accelerates atherosclerotic changes, even though the animalcontinues to produce ApoE by the liver. Fazio et al. (1997).

In atherosclerosis it is hypothesized that ApoE, via a receptor-mediatedevent, down-regulates macrophage activation in the vicinity of bloodvessel walls. Such down-regulation of macrophage activation interruptsor interferes with the cascade of events associated with atheroscleroticplaque formation, to thereby reduce or slow the formation ofatherosclerotic lesions. The cascade of events known to be associatedwith atherosclerosis includes smooth muscle cell and endothelial cellproliferation, and foam cell formation. Evidence exists that ApoEdownregulates each of these processes. ApoE thus affects the presenceand progression of atherosclerosis in vivo, independent of its effectson lipids. The progression of atherosclerosis may be assessed bymeasuring the amount or size of atherosclerotic plaques, or thepercentage of the blood vessel blocked by an atherosclerotic lesion, orthe rate of growth of such plaques.

The present inventors have for the first time demonstrated that ApoEtransduces a calcium-mediated signal (Ca²⁺/inositol triphosphate signaltransduction) in macrophage, indicating that ApoE modifies macrophagefunction by downregulating macrophage activation and, therefore,subsequent inflammation. Peptides, compounds, methods and pharmaceuticalformulations as described herein in relation to microglia and CNSdisease are accordingly useful in methods of suppressing the activationof macrophages to suppress, prevent, or slow atherosclerosis.

Atherosclerosis refers to the thickening of the arterial intima andaccumulation of lipid in artherosclerotic plaques. Administration ofcompounds of the present invention to treat or prevent atherosclerosismay be by any means discussed herein as well as other suitable methodsthat are known in the art. When using the present compounds to prevent,slow or treat atherosclerotic changes, it is apparent that they need notbe formulated to pass through the blood brain barrier. Conditions thatmay be treated by the present method include atherosclerosis of thecoronary arteries; arteries supplying the Central Nervous system, suchas carotid arteries; arteries of the peripheral circulation or thesplanchnic circulation; and renal artery disease. Administration, suchas parenteral administration, may be site-specific or into the generalblood stream.

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 Microglial Nitric Oxide Production Materials and Methods

This study examined the role of endogenous apoE in modulating microglialnitric oxide (NO) production, as measured by nitrite accumulationfollowing lipopolysaccharide (LPS) stimulation of microglia.

Culture preparation and characterization: Mixed glial cell cultures wereprepared from: (a) wildtype (C57B16; Jackson Laboratories) mouse pups;(b) ApoE deficient mutant mouse pups (ApoE-deficient mice), and (c)transgenic mouse pups expressing human ApoE3 but not murine ApoE (ApoE3mice). See Xu et al., Neurobiol. Dis. 3:229 (1996) regarding thecreation and characterization of the transgenic mice. Mixed glial cellcultures were prepared as has been described. See McMillian et al.,Neurochem. 58:1308 (1992); Laskowitz et al., J. Neuroimmunol. 76:70(1997). Briefly, brains were removed from 2-4 day old pups, cleaned ofmembranes and blood vessels, mechanically dispersed in Ca⁺²-free media,and collected by centrifugation. Cells were then plated in DMEM/F12(containing 10% fetal calf serum, 1% penicillin/streptomycin, Gibco#15070), one brain per 25 cm flask. Mixed neuronal/glial preparationswere grown in humidified incubators until confluent (3-5 weeks).

The percentage of microglia, astrocytes and neurons were quantified todemonstrate that cultures prepared from ApoE-deficient and ApoE3 micehad comparable glial populations. Immunostaining was performed usingantibodies to glial fibrillary acidic protein (GFAP; SIGMA®; 1:500dilution) and tau protein (SIGMA®; 1:500 dilution) to estimate numbersof astrocytes and neurons, and peroxidase-coupled Bandeiraeasimplifolica B4 isolectin and naphthyl acetate esterase staining wasused to detect microglia. Laskowitz et al., J. Neuroimmunol. 76:70(1997). A mixed neuronal-glial culture system was used, as this mostclosely approximates the normal CNS milieu, and allows glia-gliainteractions, which play a role in the inflammatory cascade.

Comparable glial populations were confirmed using semi-quantitativeWestern blot analysis performed for astrocytes (aGFAP; SIGMA®), neurons(atau; SIGMA®) and microglia (Bandeiraea simplifolica B4 isolectin;SIGMA®). Cellular protein was harvested at the end of experiments and 50μg protein from each sample was separated by polyacrylamide gelelectrophoresis and the protein was transferred to nylon membranes.Non-specific binding of antisera and lectin was blocked by preincubationof the membrane in 4% dried milk, 0.1% Triton X-100. Membranes wereincubated overnight with antibodies or 1 μg/ml B4 isolectin. Afterextensive washing in phosphate-buffered saline, bound antibody or lectinwas visualized by an ABC kit (Vector, Burlingame, Calif.), usingdiaminobenzidine as substrate.

Culture Stimulation Cultures were plated in serum-free media afterwashing cells once with this media, and stimulated with LPS 100 ng/ml(SIGMA®). Aliquots were taken at 24 and 60 hours for nitrite assay.

Nitrite Quantification The production of NO was assessed by measuringthe accumulation of nitrite, which was quantified using a calorimetricreaction with Griess reagent (0.1% N-1-naphthylethylenediaminedihydrochloride, 1% sulfanilamide, and 2.5% H₃PO₄). Absorbance wasmeasured at 570 nm by spectrophotometry. The sensitivity of this assayis approximately 0.5 μM.

Statistical Analysis Data were compared by ANOVA and the Fischer LSDmultiple range test; p<0.05 was considered significant.

EXAMPLE 2 Microglial Nitric Oxide Production Results

Culture Characterization: No significant differences were found in glialpopulations among the cultures prepared from ApoE-deficient, ApoE3, andwild-type mice. Cultures comprised approximately 70% astrocytes, 15%microglia and 15% neurons. Comparisons of cellular preparations fromwildtype mice, ApoE-deficient mice and ApoE3 mice showed no differencesin glial populations. In particular, levels of microglia (the primaryeffector cells for NO production) were comparable in all three culturepreparations, as detected by lectin binding (data not shown).

ApoE-deficient mouse cultures showed robust nitrite responses during thefirst 24 hours of exposure to LPS. This enhanced response was 6-foldgreater than that observed with microglia from control animals(p=0.0001; FIG. 1). Cultures from transgenic mice in which murine apoEis replaced with human ApoE3 show weak responses to LPS that were notsignificantly different than responses of wildtype animals (p=0.64 andp=0.2 at 24 and 60 hours, respectively). By 60 hours, increased nitriteaccumulation was observed in response to LPS in wildtype and ApoE3transgenic mouse preparations, although there was still a significantlygreater amount of nitrite in the apoE deficient culture as compared tocontrols (p=0.04%; FIG. 1)

The above studies show that ApoE deficient mixed neuronal-glial culturesrespond differently to LPS stimulation than glial cultures prepared frommice expressing native murine ApoE3 or those expressing the human ApoE3isoform. These results are consistent with ApoE being a biologicallyrelevant mediator of the CNS response to injury. These studiesdemonstrate that endogenous ApoE modulates glial secretion ofLPS-stimulated nitric oxide production, and suggest that one function ofendogenous ApoE produced within the brain is to suppress microglialreactivity and thus alter the CNS response to acute and chronic injury.

EXAMPLE 3 Suppression of Microglial Activation by Peptides of SEQ IDNO:3

Enriched microglia primary cultures were prepared from the brains ofapoE deficient mouse pups as described in Example 1, above. Themicroglia were stimulated with lipopolysaccharide (100 ng/ml) toactivate the microglia as described in Example 1. Activated microgliasecrete inflammatory cytokines and nitric oxide; the secretion of nitricoxide was used in the present experiment as a marker of microglialactivation. Nitric oxide production was assessed as described in Example1.

Peptides of SEQ ID NO:3 were added to cultures of activated microglia,in dosages of from 0 μM to 1000 μM. A dose-dependent decrease in nitricoxide secretion was observed after 48 hours (FIG. 2). The administrationof a peptide of SEQ ID NO:2 in a dose of 2 mM did not result in anyapparent decrease in nitric oxide secretion (FIG. 2). The monomerpeptide of SEQ ID NO:2 acted as a control to establish that the observedresults are not due to any non-specific peptide effect.

EXAMPLE 4 Effect of ApoE on Macrophage

Intracellular signaling pathways of ApoE were investigated usingperitoneal macrophage.

Thioglycolate-elicited peritoneal macrophage were harvested from 8-weekold C57-BL6 mice, and plated at a density of 4×10³ cells on glasscoverslips, loaded with 2.5 μM Fura-2/AM for thirty minutes, and washedwith Hanks buffered solution containing 75 calcium. After exposure to 5nM human recombinant apoE3 or E4, intracellular calcium was measured byZeiss digital microscopy. As shown in FIG. 3A, ApoE caused intracellularmobilization of intracellular calcium in the macrophage. Preincubationwith 100 molar excess of Receptor Associated Protein (RAP) did not blockthis effect; RAP is a physiological antagonist to LRP and blocks thefunction of LRP.

Macrophage were also plated at a density of 2×10⁶ cells/well, labeledwith ³H-myoinositol (8 μC/ml) 16 hours at 37 degrees, and exposed tohuman ApoE3 or ApoE4 (5 nM). Control cells were exposed to vehicle butnot ApoE. Results are shown in FIG. 3B; values are expressed as thepercent change in inositol trisphosphate in treated cells as compared tocontrol cells.

Exposure of peritoneal macrophage to ApoE induced a rise inintracellular calcium associated with turnover of inositoltris-phosphate (FIGS. 3A and 3B). The present results indicate that ApoEinitiates a signal transduction pathway that affects and modifiesmacrophage function. The present data suggest that ApoE downregulatesmacrophage activation and inflammation; macrophage activation andinflammation is known to contribute to the atherosclerotic process.

EXAMPLE 5 Suppression of Microglial Activation Using Peptides of SEQ IDNO:6

A 20-amino acid peptide derived from the receptor binding region ofapoE, containing amino acids 130-149 (SEQ ID NO:6) was preparedaccording to methods known in the art.

Primary murine microglial cultures were prepared as described in Example1, from apoE deficient mouse pups. In some cultures the microglia wereactivated with lipopolysaccharide (100 ng/ml), as described in Example1.

Peptides of SEQ ID NO:6 were added to cultures of activated andnon-activated microglia, in dosages of 0 μM (control), 10 μM, 100 μM and1000 μM (FIG. 4). Each dosage level of peptide was tested alone(squares) and in combination with LPS (100 ng/ml; circles). Theproduction of TNFα was then measured 24 hours after addition of thepeptides. A decrease in TNFα production by activated microglia (comparedto control culture) was observed with each peptide dose used (FIG. 4,circles). Data in FIG. 4 is presented in at least triplicate at eachdose; error bars represent standard error of the mean).

These results indicate that peptides of SEQ ID NO:6 suppress cytokinerelease from activated glial cells.

EXAMPLE 6 Cytotoxicity of Peptides of SEQ ID NO:6

The toxic effects of peptides of SEQ ID NO:6 was investigated. Culturesof activated (LPS) and non-activated microglia, as described in Example5, were used. Peptides having SEQ ID NO:6 were added to cell cultures inamounts of 0 μM (control), 10 μM, 100 μM and 1000 μM; each dosage levelof peptide was tested alone (squares) and in combination with LPS (100ng/ml; circles). Cell viability was then measured by optical density 24hours after addition of the peptides.

As shown in FIG. 5, optical density was approximately the same incultures receiving 0 μM and 10 μM of peptide, but decreased in culturesreceiving 100 μM or 1000 μM. These results, taken with the results ofExample 5, indicate that a non-toxic concentration of a peptide of SEQID NO:6 is sufficient to suppress glial cytokine release.

EXAMPLE 7 Suppression of Glial Cytokines and Cytotoxicity of Peptides ofSEQ ID NO:6

The experiments as described in Examples 5 and 6 were repeated using apeptide doses of 0 μM (control), 1 μM, 10 μM, 100 μM and 1000 μM. Eachdosage level of peptide was tested alone (squares) and in combinationwith LPS (100 ng/ml; circles). The production of TNFα was measured 24hours after administration of the peptides, and results are shown inFIG. 6. The optical density of the cell cultures was also measured (at24 hours) to assess cell viability; results are shown in FIG. 7.

These results show that microglial cytokine release was suppressed incell cultures receiving as little as 1 μM of peptide, but cytotoxiceffects were seen only in cultures receiving much larger doses ofpeptide. The results of examples 5-7 indicate that non-toxicconcentrations of peptides comprising the receptor binding region ofapoE are able to suppress cytokine release from activated microglia.

EXAMPLE 8 In Vivo Treatment of Focal Ischemia

A murine model of focal ischemia-reperfusion is used to assess theeffects of intrathecal, intravenous or intraperitoneal administration ofsmall therapeutic peptides (fewer than 30 amino acids in length)comprising the apoE LDL receptor region. One such peptide has SEQ IDNO:6.

Wild-type mice are subjected to middle cerebral artery occlusion andreperfusion according to techniques known in the art (see, e.g.,Laskowitz et al., J. Cereb. Blood Flow Metab. 17:753 (July 1997)). Onegroup of mice (wild-type control) receives no treatment after cerebralartery occlusion; in a similar group (wild-type treatment group) eachmouse receives intrathecal, intraperitoneal or intravenous injection ofa therapeutic peptide. Therapeutic peptides may be injected in varyingdoses, using the in vitro data provided above as an initial guide.

Each animal is evaluated neurologically at a predetermined time afterreperfusion (e.g., 24 hours after reperfusion) (see, e.g. Laskowitz etal., J. Cereb. Blood Flow Metab. 17:753 (July 1997)). After neurologicalexamination each mouse is anesthetized and sacrificed and the brain issectioned and stained, and infarct volume is measured. Neurologicaloutcome and infarct size is compared between control and treatmentgroups.

The above experiments may be repeated using apoE deficient mice.

EXAMPLE 9 In Vivo Treatment of Global Ischemia

A murine model of global ischemia, adapted from the rat two vesselocclusion model of global ischemia, is used to assess the effects ofintrathecal administration of small therapeutic peptides (fewer than 30amino acids in length) comprising the apoE LDL receptor region. One suchpeptide has SEQ ID NO:6.

Wild-type mice (21±1 grams) are fasted overnight, anesthetized withhalothane or another suitable anesthetic, intubated and mechanicallyventilated. The right internal jugular vein and femoral artery arecannulated. Pericranial temperature is held at 37.0 C. The carotidarteries are occluded and mean arterial pressure is reduced to 35 mmHgwith 0.3 mg intra-arterial trimethaphan and venous exsanguination. Tenminutes later ischemia is reversed. Control mice receive no additionaltreatment, test mice receive intrathecal, intravenous or intraperitonealinjection of a therapeutic peptide. Peptides may be injected at varyingdoses, using the in vitro data provided herein as a guide.

Each animal is evaluated neurologically at a predetermined time (e.g.,1, 3 or 5 days after reperfusion), using known neurological testingprocedures (see, e.g., Laskowitz et al., J. Cereb. Blood Flow Metab.17:753 (July 1997)). After neurological evaluation, each animal isanesthetized and sacrificed and the brain injury is assessed usingmethods known in the art.

For example, brains may be perfusion fixed in situ, then sectioned,stained and examined by light microscopy, for example, to determineinjury to the CA 1 sector of the hippocampus, and viable and non-viableneurons counted and compared.

Neurological outcome and brain injury is compared between control andtreatment groups.

EXAMPLE 10 Apolipoprotein E and ApoE-Mimetic Peptides Initiate aCalcium-Dependent Signaling Response in Macrophages

This example shows that apoE initiates a signaling cascade in murineperitoneal macrophage that is associated with mobilization ofintracellular Ca²⁺ stores following increased production of inositoltrisphosphate. This cascade was inhibited by pretreatment withreceptor-associated protein and Ni²⁺. Signal transduction was mediatedby a pertussis toxin-sensitive G protein. These are characteristicproperties of signal transduction induced via ligand binding to thelipoprotein receptor-related protein (LRP) receptor. A peptide derivedfrom the receptor binding region of apoE also initiated signaltransduction in the same manner as the intact protein. The presence ofcross desensitization suggested that the apoE and the apoE-mimeticpeptide competed for the same binding site. This was confirmed by ourobservation that radiolabeled apoE-mimetic peptide competed with theintact protein for receptor binding. These data indicates thatApoE-dependent signal transduction mediates the immunomodulatoryproperties of this lipoprotein.

Materials and Methods

Materials. Brewer's thioglycollate broth was purchased from DifcoLaboratories (Baltimore, Md.). RPMI Medium 1640, fetal bovine serum,Hanks' Balanced Salt Solution and other cell culture reagents werepurchased from Life Technologies, Inc. (Grand Island, N.Y.). Bovineserum albumin (BSA), pertussis toxin, and HEPES were from Sigma ChemicalCo. (St. Louis, Mo.). Fura-2AM and BAPTA/AM were obtained from MolecularProbes (Eugene, Oreg.). Myo-[2-³H]inositol (specific activity 10-20Ci/mmol) was purchased from American Radiolabeled Biochemicals (St.Louis. Mo.). A plasmid containing the RAP cDNA was a kind gift from Dr.Joachim Herz, the University of Texas, Southwestern, Dallas Tex. It wasused to produce RAP as previously described [21]. Human recombinantapoE2 was obtained commercially from Panvera Corp (Madison, Wis.). Thepreparation was free of endotoxin, and homogenous as judged bySDS-polyacrylamide gel electrophoresis. [³H]thymidine (specificactivity, 70 Ci/mmol) and Iodine-125 (specific activity: 440 mCi/mg)were purchased from the American Radiolabeled Chemicals, Inc. (St. LouisMo.). The 20 amino acid ApoE mimetic peptide(Ac-TEELRVRLASHLRKLRKRLL-amide) with and without a tyrosine on the aminoterminus as well as a scrambled control peptide of identical size, aminoacid composition, and purity were synthesized by QCB Biochemicals(Hopkinton, Mass.) to a purity of 95%. All amino termini were acetylatedand all carboxyl termini were blocked with an amide moiety. Peptideswere reconstituted in sterile isotonic phosphate buffered saline. Ascrambled control peptide of identical size, amino acid composition, andpurity was also synthesized. All other reagents used were of the highestquality commercially available.

Macrophage Harvesting. All experiments involving animals were firstapproved by the Duke Institutional Animal Care and Use Committee.Pathogen-free female C57BL/6 mice and ApoE deficient mice previouslybackcrossed 10 times to the C57BL/6 strain were obtained from theJackson Laboratory (Bar Harbor, Me.). Thioglycollate-elicited peritonealmacrophages were harvested by peritoneal lavage using 10 ml of ice-coldHanks' balanced salt solution containing 10 mM HEPES and 3.5 mM NaHCO₃(HHBSS), pH 7.4. The macrophages were pelleted by centrifugation at 4°C. at ˜800×g for 10 min and resuspended in RPMI 1640 media supplementedwith 25 mM HEPES, 12.5 U/ml penicillin, 6.5 mg/ml streptomycin, and 5%fetal bovine serum. Cell viability was determined by the trypan blueexclusion method and was consistently greater then 95%.

Receptor Binding Studies. Macrophages were plated in 48-well cellculture plates (Costar) at 2.5×10⁵ cells per well and incubated for 3 hat 37° C. in a humidified 5% CO₂ incubator. The plates were then cooledto 4° C. and unbound cells were removed by three consecutive rinses withice-cold Hanks' balanced salt solution containing 20 mM Hepes and 5%BSA, pH 7.4 (binding buffer). To quantify direct binding of the¹²⁵I-apoE mimetic peptide, varying amounts of radiolabeled peptide wereadded to each well in the presence or absence of 200-fold molar excessof unlabeled peptide. Specific binding to cells was determined bysubtracting the amount of ¹²⁵I-apoE peptide bound in the presence ofexcess unlabeled peptide (nonspecific binding) from the amount of¹²⁵I-apoE peptide bound in the absence of excess unlabeled peptide(total binding). For competition studies, 50 nM radiolabeled peptide wasadded to each well in the presence or absence of varying amounts (31.25nM-4 μM) of unlabeled ApoE2 or P. Cells were then incubated at 4° C. for12-16 h. Unbound ligand was removed from the wells and the cellmonolayer was rinsed three times with ice-cold binding buffer. Cellswere then solubilized with 1 M NaOH, 0.5% SDS at room temperature for >5h before the contents of each well was added to polystyrene tubes andcounted in a LKB-Wallac, CliniGamma 1272 γ-counter (Finland).

Measurement of [Ca²⁺]_(i) in apoE and peptide treated macrophage.Changes in [Ca²⁺]_(i) levels in Fura-2/AM treated single cells werequantified using digital imaging microscopy in accordance with knowntechniques. Macrophages were plated on glass coverslips sitting in 35 mmPetri dishes at a density of 1.5×10⁵ cells/cm², and allowed to adherefor 2 h in a humidified 5% CO₂ incubator at 37° C. The non-adherentcells were aspirated and the monolayers were washed twice with HHBSS. 4μM Fura-2/AM was incubated with the cells for thirty min in the dark atroom temperature and [Ca²⁺]_(i) was subsequently measured using adigital imaging microscope in accordance with known techniques. Afterobtaining baseline measurements for 5 min, ligand (apoE, apoE mimeticpeptide, or scrambled peptide) was added, and multiple [Ca²⁺]_(i)measurements were taken. To determine if signaling resulted fromligation of the ligand to LRP, cells were preincubated with a 1000-foldmolar excess of RAP or 10 mM NiCl₂, both of which inhibit ligand bindingto LRP, for 5 min prior to stimulation with apoE or peptide. Inexperiments in which the involvement of a G protein was assessed,monolayers were incubated with 1 μg/ml pertussis toxin for 12 h at 37°C. and Ca²⁺ measurements were made as stated above.

Measurement of IP₃ in apoE treated macrophage and effect of pertussistoxin. The formation of IP₃ in myo-[2-³H]inositol-labeled macrophagesunder various experimental conditions was quantified in accordance withknown techniques. Macrophage were plated in 6 well plates (4×10⁶cells/well) and allowed to adhere at 37° C. for 2 h in a humidified 5%CO₂ incubator. Medium was aspirated from the monolayers and RPMI 1640medium containing 0.25% BSA and myo-[2-³H]inositol (specific activity10-20 Ci/mmol) was added to each well. The cells were incubated at 37 (Cfor an additional 16-18 h. Monolayers were rinsed three times with 25 mMHHBSS containing 1 mM CaCl₂, 1 mM MgCl₂, 10 mM LiCl, pH 7.4. A volume of0.5 ml of this solution was added to each well, and the cells werepreincubated for 3 min at 37° C. before stimulated with ligand. Thereaction was stopped by aspirating the medium containing the ligand andadding 6.25% perchloric acid. The cells were scraped out of the wells,transferred to tubes containing 1 ml of octylamine/Freon (1:1 vol/vol)and 5 mM EDTA, and were centrifuged at 5600×g for 20 min at 4° C. Theupper phase solution was applied to a 1 ml Dowex resin column (AG 1-X8formate; Bio Rad Laboratories, Richmond, Calif.) and eluted sequentiallyin batch process with H₂O, 50, 200, 400, 800, and 1200 mM ammoniumformate containing 0.1 M formic acid [26]. Radioactivity was determinedby placing aliquots in a liquid scintillation counter to determineradioactivity. To evaluate the pertussis-toxin sensitivity of the Gprotein coupled to receptor activation and phosphatidyl inositol4,5-bisphosphate (PIP₂) hydrolysis, cells were plated as described aboveand incubated with μg/ml pertussis toxin which had been preactivatedwith 40 mM DTT at 30° C. for 20 min. The effect on IP₃ formation wasmeasured as described above.

Competition between apoE and apoE mimetic peptide for binding site onthe receptor. Changes in macrophage [Ca²⁺]_(i) upon stimulation withapoE and apoE-mimetic peptide were studied to determine whether theseligands bind to the same receptor. Fura-2/AM loaded macrophages wereincubated overnight, plated on glass cover slips, stimulated with oneligand, and changes in [Ca²⁺]_(i) quantified. Cells were then stimulatedwith second ligand and Ca²⁺ measurements repeated.

Results

Effect of apoE on macrophage [Ca²⁺]_(i). Modulation of free cytoplasmicCa²⁺ concentration is a ubiquitous signaling response. In many celltypes, binding of ligands to plasma membrane receptors activates thehydrolysis of PIP₂ by membrane-bound phospholipase C, generating IP₃.IP₃ causes the release of Ca²⁺ from the endoplasmic reticulum by bindingto its cognate receptor, which is also a Ca²⁺ channel. In non-excitablecells, [Ca²⁺]_(i) signaling is associated both with Ca²⁺ release fromintracellular stores and Ca²⁺ influx. Treatment of macrophages withhuman recombinant apoE increased [Ca²⁺]_(i) levels 2-4-fold compared tomacrophage treated with buffer (FIG. 8A). In a typical experiment[Ca²⁺]_(i) levels in unstimulated cells and apoE-treated cells were95.33±7.37 and 180.25±14.57 nM, respectively. The increase in [Ca²⁺]_(i)upon stimulation with apoE was observed in 70-80% of the cells examined.ApoE-induced increase in [Ca²⁺]_(i) was heterogeneous, asynchronous, andeither oscillatory or sustained. ApoE-induced increases in macrophage[Ca²⁺]_(i) was dose-dependent (FIG. 8B). To address the possibility thatnative apoE secreted by macrophage altered responses to exogenous humanrecombinant apoE, these experiments were repeated using macrophageprepared from apoE deficient mice. Calcium responses followingstimulation with apoE were identical in wild-type macrophages andmacrophages from apoE deficient mice (data not shown).

The effect of pertussis toxin on apoE-induced IP₃ synthesis. Exposure ofmyo-[2-³H] inositol-labeled macrophage to apoE caused a 1.5-2.0-foldincrease in IP₃ levels (FIG. 9A). This effect was dose-dependent (FIG.9B). Pretreatment of the macrophages with pertussis toxin completelyabolished this increase in IP₃. (FIG. 9A). These studies demonstratethat the phospholipase C-catalyzed hydrolysis of membrane PIP₂ in apoEstimulated cells is coupled to a pertussis toxin-sensitive G protein.

ApoE-induced increases in macrophage [Ca²⁺]_(i) are attenuated by Ni²⁺and Rap. Previous studies have demonstrated that ApoE binds to LRP andis then internalized. Additionally, binding of lactoferrin, Pseudomonasexotoxin A, lipoprotein lipase and thrombospondin to LRP initiates asignaling cascade associated with the generation of second messengers.To investigate the possibility that LRP is involved in the signalcascade induced by apoE, macrophages were preincubated with RAP and Ni⁺²prior to stimulation with apoE2 or apoE2 mimetic peptide. RAP is a 39 kDprotein that blocks the binding of all known ligands to LRP. Ni²⁺ alsoblocks ligand interactions with LRP. Both preincubation with RAP andNi⁺² markedly attenuated the [Ca²⁺]_(i) increases associated withsubsequent exposure to apoE (data not shown). These results areconsistent with the hypothesis that apoE induces a signaling cascade viaspecific interaction with LRP. Pretreatment of macrophage with pertussistoxin also markedly attenuated the ApoE-dependent Ca²⁺ response,indicating that signal transduction induced by apoE is coupled to apertussis toxin-sensitive G protein. This is consistent with the knownproperties of LRP-dependent signal transduction.

Effect of apoE-mimetic peptide on macrophage [Ca²⁺]_(i). Stimulation ofmacrophage with the peptide derived from residues 130-149 of the apoEreceptor binding region also resulted in a 2-3-fold increase in[Ca²⁺]_(i) whereas a scrambled control peptide of identical size andcomposition had no effect (data not shown). This increase in [Ca²⁺]_(i)was observed in approximately 60-70% of cells examined. As with the apoEresponses, peptide-induced increases in macrophage [Ca²⁺]_(i) wereheterogeneous and asynchronous. These results demonstrate that bothintact apoE and a peptide derive from the apoE receptor binding regioninduce an increase in [Ca²⁺]_(i) that is consistent with the initiationof a signaling cascade. However, on a molar basis, higher concentrationsof peptide were necessary to get [Ca²⁺]_(i) responses compared to theintact apoE. This difference likely results from differences in receptoraffinity between the peptide and apoE, a property generally seen whencomparing the effects of intact proteins to peptide ligands.

Effects of repeated stimulation of apoE and apoE-mimetic peptide on[Ca²⁺]_(i). We evaluated the possibility of competition between apoE andits mimetic peptide for binding sites on the receptor by quantifying thechanges in [Ca²⁺]_(i) consequent to receptor ligation. Followingrepeated exposure to apoE, there was a marked attenuation in [Ca²⁺]_(i)suggesting tachyphylaxis (data not shown). Following the increase in[Ca²⁺]_(i) associated with the initial exposure to human recombinantapoE, there was a marked attenuation in [Ca²⁺]_(i) response tosubsequent peptide exposure (data not shown). Similarly, there was aloss of [Ca²⁺]_(i) response to apoE addition following initial exposureto peptide (data not shown). No desensitization in calcium response wasobserved with exposure of scrambled peptide (data not shown). Thisobserved tachyphylaxis suggests receptor desensitization secondary toreceptor ligation, and is consistent with the hypothesis that both theintact apoE protein and the 20 residue peptide bind to the samereceptor.

Discussion

The primary observations of this example are that: 1) binding toreceptors on the macrophage cell surface of human recombinant apoE (inpM to nM concentrations) initiates signaling events associated withincreases in [Ca²⁺]_(i) and IP₃; 2) a 20 residue peptide derived fromthe receptor binding region of apoE, but not a scrambled controlpeptide, causes identical changes in macrophage [Ca²⁺]_(i); 3) changesin [Ca²⁺]_(i) and IP₃ are specific and dose-dependent; 4) apoE-inducedincrease in cellular IP₃ is pertussis toxin-sensitive; and 5) changes in[Ca²⁺]_(i) are blocked by RAP and Ni²⁺. Moreover, based on the presenceof cross-desensitization, apoE and the apoE-mimetic peptide appear tobind to the same receptor.

EXAMPLE 11 An Apolipoprotein E Mimetic Peptide is Protective in a MurineHead Injury Model

This Example demonstrates a protective effect of intravenousadministration of a 17 amino acid ApoE mimetic peptide (the fragment ofApoE containing amino acids 133-149 (SEQ ID NO: 10)) following headinjury.

Mice were endotracheally intubated and their lungs were mechanicallyventilated with 1.6% isoflurane at 30% partial pressure of oxygen. Themice received a midline closed head injury delivered by a pneumaticimpactor at a speed of 6.8 m/s. Thirty minutes after closed head injury,mice were randomized into 3 groups (n=16 mice per group as follows: highdose peptide (406 ug/kg), low dose peptide (203 ug/kg), and salinecontrol solution. All peptide solutions were prepared in sterileisotonic saline (100 ul) and delivered intravenously via tail veininjection. Rotorod time and weight were measured for five consecutivedays after injury. At 21 days, the ability to learn to find a hiddenplatform in the Morris Water Maze was tested.

Prior to injury, rotorod latency and weights were comparable in allanimals. After injury, the saline injected animals had a profounddeficit in rotorod testing which was associated with weight loss. Highdose peptide, and to a lesser extent low dose peptide protected animalsfrom this motor deficit (FIG. 10A), and concomitant weight loss (FIG. 10b). This protective effect of the single dose of peptide was sustainedfor five days following injury (p<0.05 3-way repeat measures ANOVA).

In addition, the peptide appeared to provide protection in learningdeficits in learning to find a hidden platform (FIG. 10C) in the MorrisWater Maze (p<0.05 3-way repeat measures ANOVA). Treatment with thepeptide also resulted in a significant improvement in acute survival asdemonstrated by Kaplan-Meier analysis (data not shown).

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is describedby the following claims, with equivalents of the claims to be includedtherein.

EXAMPLE 12 Protective Effect of Apolipoprotein E-Mimetic Peptides onN-Methyl-D-Aspartate Excitotoxicity in Primary Rat Neuronal/Glial CellCultures

The present inventors hypothesized that one mechanism by which apoEmight play a role in modifying the response of brain to ischemia is byprotecting against glutamate excitotoxicity. Glutamate is believed tocontribute to neuronal injury in the setting of ischemia. Of thedifferent classes of glutamate-activated channels, specific activationof the N-methyl-D-aspartate (NMDA) receptor is believed to be primarilyresponsible for mediating calcium influx and exacerbation of neuronalinjury in a variety of neuron types (Meldrum and Garthwaite, 1990).

To model the in vivo effects of ApoE in an experimental setting ofcerebral ischemia, we examined the effects of biologically relevantconcentrations of native human ApoE and peptides derived from thereceptor-binding region of ApoE in a cell culture model of primary ratneocortical neurons and glia exposed to NMDA. Intact ApoE exhibited amodest dose-dependent reduction in NMDA induced cytotoxicity. Bycomparison, a seventeen residue ApoE-mimetic peptide exhibited enhancedneuroprotection relative to native ApoE and completely blocked both thecalcium influx and cell death associated with NMDA exposure. Furthertruncation of the peptide at the amino terminus resulted in aprogressive loss of neuroprotection from NMDA excitotoxicity. Theseresults suggest that ApoE affects recovery of neuronal cells fromischemic injury following brain insult by protecting cells againstglutamate toxicity. Furthermore, they implicate the use of ApoE-mimeticpeptides as a therapeutic strategy following cerebral ischemia and otherdiseases and disorders associated with glutamate toxicity.

Experimental Procedures

All animal procedures were designed to minimize animal discomfort andnumbers, and were approved by the Duke University Animal Care and UseCommittee.

Preparation of Primary Neuronal-Glial Cultures

Primary neuronal-glial cultures were prepared from fetal Sprague-Dawleyrat brains at 18 days of gestation as previously described (Pearlsteinet al., 1998). Brains were harvested from 10-15 pups and dissected toseparate cortex from meninges and subcortical structures usinganatomical landmarks. Cortices were pooled and minced into 2 mm³ piecesin a buffered salt solution supplemented with 20 mM HEPES buffer, pH7.4, containing 0.25% trypsin. The tissue was incubated for 20 minutesat 37° C. in a 5% CO₂/95% room air atmosphere, and washed twice withice-cold, glutamine-free minimum essential medium (MEM; LifeTechnologies) containing 15 mM glucose, 5% fetal bovine serum (GIBCO),5% horse serum (GIBCO), and 1% DNase-I (Sigma Chemical Co., St. Louis,Mo., USA). Tissue pieces were dissociated by trituration through afire-polished 9-inch Pasteur pipette (I.D.=0.7 mm). The resultantsuspension was centrifuged at 50×g for 10 min, the supernatantdiscarded, and the pellet resuspended in growth medium (MEM supplementedwith 15 mM glucose, 5% fetal bovine serum, and 5% horse serum). Thedissociated cells were plated to achieve a confluent monolayer using4×10⁵ cells per well in poly-D-lysine coated, 24-well culture plates(Falcon 3047; Becton Dickinson Co., Lincoln Park, N.J., USA). Cultureswere maintained undisturbed at 37° C. in a humidified 5% CO₂/95% roomair atmosphere for 13-16 days prior to use. Cultures prepared accordingto this protocol were found to contain 54% neurons and 46% glia asdetermined by immunohistochemical staining for NF-160 and glialfibrillary acidic protein (Kudo et al., 2001).

Synthesis of Apo-E Peptides

Peptides were synthesized by QCB Biochemicals (Hopkinton, Mass.) to apurity of 95% and reconstituted in sterile isotonic phosphate bufferedsaline (PBS). For each peptide, the amino terminus was acetylated, andthe carboxyl terminus was blocked with an amide moiety. The parent17-residue peptide ApoE (133-149) (SEQ ID NO:10) was derived from thereceptor-binding region of apoE. A scrambled control peptide ofidentical size, amino acid composition, and purity(Ac-LARKLRSRLVHLRLKLR-amide) (SEQ ID NO:14) was similarly created. The14-residue sequence (136-149) and 11-residue sequence (139-149) (SEQ IDNO:11) were made by progressively truncating the amino terminus of theparent (133-149) (SEQ ID NO:12) peptide.

Exposure to NMDA/Cytotoxicity Assessment

Mature cultures (13-16 days in vitro) were washed with Mg²⁺-freebuffered salt solution (BSS) containing 20 mM HEPES buffer (pH7.4) and1.8 mM CaCl₂, prior to the addition of NMDA. Following NMDA exposure,cultures were maintained for 30 minutes at 37° C. in a 5% CO₂/95% airatmosphere. The medium containing NMDA was then removed and replacedwith MEM supplemented with 20 mM glucose. The cultures were returned tothe incubator for 24 h. In all experiments, cellular damage was assessedat 24 h after exposure to NMDA by measurement of the activity of lactatedehydrogenase (LDH) released into the medium as described below. Thenon-competitive NMDA receptor antagonist, MK-801 (Tocris Cookson Ins.,St. Louis, Mo.) at a concentration of 10 μM was used as a positivecontrol. Preliminary dose-response studies were performed to determinethe concentration of NMDA (100 μM) required to effect a near-maximal LDHrelease (ED₉₀) as used in the present study.

Assessing Effect of apoE and apoE-Mimetic Peptide on NMDA Toxicity

The effect of human recombinant apoE (Panverra, Madison, Wis.) on 100 μMNMDA-induced LDH release was assessed and the dose-response curve wasobtained (apoE: 0.1-10 μM final concentration). ApoE3, the most commonhuman isoform, was added to the cultures 30 minutes prior to and removedfollowing NMDA exposure. The effect of apoE peptide (133-149) on NMDA(100 μM or 300 μM)-induced LDH release was next assessed and thedose-response curve was obtained in a separate set of sister culturesthat were simultaneously treated under one of the following conditions:(1) various doses of peptide (0.3 μM, 1 μM, 3 μM, 6 μM, 10 μM finalconcentration) were added to the culture immediately prior to andremoved at the end of NMDA exposure; (2) no peptide, NMDA exposure; (3)no peptide, no NMDA exposure; (4) no peptide, NMDA exposure with 10 μMMK-801 for 30 minutes. The effect of a scrambled control peptide onNMDA-induced LDH release was similarly assessed.

The effect of time of administration of apoE peptide (133-149) onNMDA-induced LDH release was assessed in a separate set of sistercultures that were simultaneously treated under one of followingconditions: (1) peptide was added to the culture medium 24 h prior toand removed immediately before exposure to NMDA; (2) peptide was addedimmediately prior to and removed at the end of the NMDA exposure; (3)peptide was added immediately following the 30 minutes NMDA exposure;(4) no peptide, NMDA exposure; (5) no peptide, no NMDA exposure. In allcases, peptide concentration was 6 μM, cultures were exposed to 100 μMNMDA for 30 minutes at 37° C., and the effect of treatment/exposure wasexamined 24 h after NMDA exposure as described above.

Measurement of LDH Release

Cellular injury was quantitatively assessed 24 h after excitotoxicstress by measuring the amount of lactate dehydrogenase (LDH) releasedinto overlying medium by damaged cells. LDH activity was determined by amodification of methods previously described (Amador et al., 1963). Inbrief, a 200 μl sample of culture medium was added to a polystyrenecuvette containing 10 mM lactate and 5 μmol of NAD in 2.75 ml of 50 mMglycine buffer (pH 9.2) at 24° C. LDH activity was determined from theinitial rate of reduction of NAD as calculated using a linear leastsquare curve fit of the temporal changes in fluorescence signal from thecuvette (340 nm excitation, 450 nm emission) and expressed in units ofenzymatic activity (nmol of lactate converted to pyruvate per min).Analysis was performed on a fluorescence spectrophotometer (Perkin ElmerModel LS50B; Bodenseewerk GmbH, Uberlinger, Germany).

Effect of Peptides on Cellular Calcium Uptake

Cellular calcium uptake from the extracellular space was assessed using⁴⁵CaCl₂ (American Radiolabeled Chemicals, St. Louis, Mo.). After washingthe cultures with Mg²⁺-free BSS containing 20 mM HEPES buffer, 6 μMpeptide or 10 μM MK-801 was added to each well prior to and removed atthe end of 100 μM NMDA exposure. ⁴⁵Ca (0.28 μCi/ml, 0.9 μCi/well) wasadded to each well immediately prior to NMDA exposure. The cultures werereturned to the incubator and maintained at 37° C. Twenty minutes later,the exposure medium was removed and each well was washed 3 times withice-cold Mg²⁺-free BSS containing 20 mM HEPES buffer. The cells weresubsequently lysed by addition of 0.2% sodium dodecyl sulfate (SDS). Analiquot from each well was added to a liquid scintillation vialcontaining 10 ml Cytoscint™ (ICN, Biochemical Research Product, CA) andradioactivity was determined by scintillation counting and normalized tocell count.

Circular Dichroism

Circular dichroism spectra were recorded on an Aviv Model 202 circulardichroism spectrometer, using 1 mm pathlength quartz cuvettes. Peptideconcentration was approximately 50 μM in a buffer of PBS. Spectra weretaken at 273 K. Samples contained 50 μM peptide in phosphate-bufferedsaline (PBS) and spectra were recorded at 4° C. Percent helicities werecalculated from the signal at 222 nm using the equation as previouslydescribed (Myers et al., 1997). Concentrations of peptide stocksolutions were determined by quantitative amino-acid analysis, carriedout by the Protein/DNA technology Center at the Rockefeller University.

Statistical Analysis

Multiple group comparisons were performed by one-way analysis ofvariance. When comparisons to single control group were needed, post hocanalysis was performed using Dunnet's test. Values are reported asmean±standard deviation. Significance was assumed when P<0.05.

Results

To investigate the ability of ApoE to protect cells from glutamateexcitotoxicity in an experimental tissue culture model of cerebralischemia, primary rat neuronal/glial cultures were preincubated withhuman recombinant ApoE prior to exposure of the cells to NMDA. Previousexperiments performed in our laboratory failed to detect an isoformspecific effect of human recombinant ApoE on NMDA-induced excitotoxicityin primary rodent neuronal/glial cultures (Aono et al, 2002). To thisend ApoE3, the most common isoform (Corder et al, 1993), was usedthroughout the experiments described herein.

To determine the dose-response of ApoE3 on NMDA-induced cell damage,primary cultures were first preincubated with varying concentrations ofapoE3 for 30 minutes prior to exposure with 100 μM NMDA (FIG. 11).Cellular injury was assayed 24 h following excitotoxic stress bymeasuring the amount of LDH released by damaged cells into the media(LDH release=nmols of lactate converted to pyruvate per min; seeExperimental Procedures). Exposure of cultures to NMDA alone caused asignificant increase in LDH release (2.82+/−0.19) compared to controluntreated cells (1.01+/−0.06; p<0.05). Preincubation of the cultureswith apoE3 provided a modest, dose-dependent reduction in LDH releasewhich was significant at ApoE concentrations of 1-10 μM relative to NMDAalone (p<0.05, ANOVA followed by Dunnett's test). Hence, ApoE3 confersmodest yet significant neuroprotection of primary rat neuronal/glialcultures from glutamate excitotoxicity.

To test the hypothesis that small ApoE-mimetic peptides could similarlyprotect neuronal/glial cultures from NMDA excitotoxicity, we created apanel of three truncated apoE peptides derived from the receptor-bindingregion of apoE (FIG. 12A; ApoE 133-149, 136-149, 139-149). To assess thestructural characteristics of the peptides and their helical content, wefirst carried out circular dichroism (CD) experiments (FIG. 12B). The CDspectra of the three peptides demonstrated evidence of significanthelical structure, as evidenced by the significant minima at 222 nm and208 nm. From these data we calculate that the three peptides have acomparable degree of helicity, with a 12-14% helical population insolution. Previous sedimentation equilibrium experiments performed inour laboratory demonstrate that these peptides exist as monomers insolution, indicating that the observed helical structure is not due toself-association of the peptide into helical oligomers (Laskowitz etal., 2001).

We first investigated the effect of the peptide ApoE (133-149) onNMDA-induced cell damage in primary neuronal/glial cultures (FIG. 13).This peptide was previously shown by our group to possess a bioactivitycapable of modulating murine microglial function (Laskowitz et al,2001). Primary rat neuronal/glial cultures were first preincubated withApoE (133-149) 30 minutes prior to NMDA exposure and LDH release wasassayed 24 h later. Exposure of cultures to 100 μM NMDA in the absenceof ApoE (133-149) caused a two-fold increase in LDH release(1.74+/−0.06) relative to unexposed control cells (0.88+/−0.10; p<0.05)(FIG. 3A). In contrast to intact ApoE3 which was only modestlyprotective, addition of the 17-residue ApoE peptide provided robust,dose-dependent protection against 100 μM NMDA toxicity.

Protection from glutamate toxicity was first observed using a 3 μm ApoE(133-149) peptide concentration with maximal protection observed at 6 μMapoE (133-149) (p<0.05, ANOVA followed by Dunnett's test). As expected,no protection was observed in the presence of the scrambled controlpeptide. Surprisingly, treatment of cultures with apoE (133-149) failedto protect cells at any peptide concentration when cultures were exposedto 300 μM NMDA exposure (FIG. 13B). These results demonstrate that anApoE mimetic peptide comprising 17 amino acid residues derived from thereceptor-binding domain of ApoE is neuroprotective followingNMDA-induced excitotoxicity. Furthermore, the peptide conferred greaterprotection to cultures than the intact holoprotein.

To further define the peptide domain required for neuroprotection in ourprimary culture system, we compared the effects of truncated ApoEpeptides on NMDA-induced cell damage (FIG. 14). Removal of the threeamino-terminal residues from the 17 residue peptide generated ApoE(136-149), a 14 amino acid peptide (FIG. 12A). Treatment with 6 μM ApoE(136-149) conferred modest protection against NMDA toxicity relative tothe 17-residue parent peptide, ApoE (133-149), which completely blocked100 μM NMDA-induced cell death (FIG. 14). By contrast, deletion of threeadditional amino terminal residues resulted in an 11-residue peptide,ApoE (139-149), which possessed no detectable bioactivity (ANOVAfollowed by Dunnett's test; p<0.05). These results indicate thattruncations from the amino terminus of the 17 residue peptide ApoE(133-149), resulted in a progressive loss of neuroprotection againstNMDA excitotoxicity, and that the amino acid domain ApoE (133-136) isnecessary for the ApoE peptide to retain bioactivity.

To investigate whether the parent ApoE-mimetic peptide exerted itsprotective effects by modulating calcium influx associated with NMDAexposure, we measured calcium uptake following incubation of the cellswith ApoE (133-149) and 100 μM NMDA (FIG. 15). Calcium uptake wasmeasured twenty minutes following exposure of the cultures to ⁴⁵Ca⁺⁺(see Experimental Procedures). Exposure of the cultures to 6 μM ApoE(133-149) in the absence of NMDA served as a control and had no directeffect on calcium influx (FIG. 15). Exposure of cultures to 100 μM NMDAin the absence of peptide induced calcium influx which was completelyreversed by pretreatment with 10 μM MK-801 (FIG. 15). Treatment with 6μM ApoE (133-149) significantly decreased calcium influx compared toNMDA alone, whereas treatment with 6 μM scrambled control peptide had noeffect on calcium influx (ANOVA followed by Dunnett's test; p<0.05).Hence, a 17-residue peptide derived from the receptor-binding domain ofapoE is capable of protecting cells from the detrimental effects ofcalcium influx associated with NMDA-induced excitotoxicity.

We next wished to examine the temporal relationship betweenadministration of the ApoE-mimetic peptide and protection following NMDAexposure. To this end, ApoE (133-149) was added to cells either 24 hoursprior to NMDA exposure, concurrently with NMDA, or post-NMDA exposure(FIG. 16; see Experimental Procedures). As before, a robust protectionwas observed when 6 μM ApoE (133-149) was administered concurrent with100 μM NMDA (FIG. 16). Pretreatment of cultures with peptide 24 hoursprior to NMDA exposure provided a modest but significant protection,whereas addition of ApoE (133-149) following NMDA exposure provided noprotection. Thus, although ApoE (133-149) afforded modest protectionwhen added 24 hours prior to NMDA exposure, administration of thepeptide concurrently provided the best protection (ANOVA followed byDunnett's test; p<0.05). Together, these results demonstrate that amonomeric peptide comprised of ApoE residues 133-149 can protect cellsfrom glutamate excitotoxicity in an experimental tissue culture model ofcerebral ischemia.

C. Discussion

In this study, we demonstrate that peptide sequences derived from thereceptor-binding region of ApoE exert a protective effect againstNMDA-mediated neuronal excitotoxicity in a tissue culture model ofcerebral ischemia. This neuroprotective effect of the ApoE peptide wasboth specific and dose-dependent. At a concentration of 6 μM, aseventeen residue peptide, ApoE (133-149) blocked the neurotoxicity andcalcium influx associated with exposure of primary neuronal-glialcultures to 100 μM NMDA as completely as the NMDA receptor antagonistMK-801.

Although there are multiple clinical reports demonstrating that apoEgenotype influences neurological recovery in isoform-specific fashion,the mechanisms by which this occur remain poorly defined. It has beenproposed that endogenbus apoE may influence the CNS response to injuryby modifying oxidative stress (Miyata and Smith, 1996), exerting directneurotrophic effects (Holtzman et al., 1995), downregulating the CNSinflammatory response (Lynch et al., 2001), or serving as a pathologicalchaperone by promoting cerebral amyloid deposition (Wisniewski andFrangione, 1992). In this report, we found that the intact ApoE proteinalso confers a modest degree of neuroprotection from NMDA-inducedtoxicity. This is in contrast with other recent studies, which failed todemonstrate any neuroprotective effect from the intact ApoE protein(Jordan et al., 1998; Lendon et al., 2000). These discordant results mayin part be due to differences in methodology and experimental design.For example, Lendon et al. found that a 5 μg/ml concentration of ApoEfailed to demonstrate additional neuroprotection beyond that of HDLalone, which itself conferred a modest benefit against NMDA-inducedneurotoxicity. Unfortunately, in that study the effects of ApoE alonewere not studied (Lendon et al. 2000). Jordan et al. also failed todemonstrate neuroprotection from ApoE in a model of NMDA-inducedneurotoxicity. It is worth noting, however, that in these experiments,ApoE from conditioned media was used rather than human recombinant ApoE,and ApoE was preincubated for 5 days prior to NMDA exposure, as comparedto 30 minutes in our study.

Our results suggest that biologically relevant concentrations of ApoEconfer a modest degree of neuroprotection from excitotoxic cell death.Interestingly, the peptide derived from the receptor binding region ofApoE exerted a much more robust neuroprotective effect than the intactholoprotein, and completely blocked the cell death and calcium influxassociated with the exposure of neocortical neurons to 100 μM NMDA. Oneexplanation for our observations is that ApoE and the peptide fragmentsbound to the NMDA receptor. Although competitive antagonism of the NMDAreceptor might explain the loss of neuroprotection of the peptide athigher excitotoxic burdens, competition at the NMDA receptor has neverbeen demonstrated either for ApoE or for peptide fragments derived fromApoE.

Another plausible explanation is that these peptides exerted theirbiological activity against NMDA toxicity indirectly by interacting withspecific cell surface receptors in the same manner as the intact ApoEholoprotein. ApoE is known to bind a family of cell surface receptors,including the LDL, VLDL, LRP/α2M, ER-2, and LR8 receptors (Kim et al.,1996; Novak et al., 1996). One region of ApoE which is critical for theinteraction with the LDL receptor lies between residues 140-160 (Mahley,1988), and site-specific mutagenesis studies of this region havedemonstrated that mutations affecting charge and conformation can resultin defective binding (Lalazar, 1988). We have previously demonstratedthat the peptides derived from the receptor binding region identical tothose used in this study compete with ApoE for receptor binding (Misraet al., 2001). In fact, ApoE has been demonstrated to initiate asignaling cascade in both neurons and macrophage (Misra et al., 2001;Muller et al., 1998).

A recent report has demonstrated that the LRP receptor is capable ofinitiating a calcium signaling response mediated by the NMDA receptor(Bacskai et al., 2000). Although the exact mechanism by which thisoccurs remains undefined, the authors speculate on the presence of aneuron-specific intracellular adaptor protein that modulates NMDAactivity following ligand binding and dimerization of the LRP receptor,and this conceivably could lead to a protective response downstream fromthe NMDA receptor. Interestingly, recent observations have suggestedthat the presence of receptor-associated protein (RAP), which blocks allknown LRP interactions, does not reverse the neuroprotective effects ofApoE in a cell culture paradigm of NMDA excitotoxicity (Aono et al.,2002).

In the native holoprotein, the receptor binding region is in an ahelical conformation. To confirm that the peptide fragments used in ourstudies were capable of adopting this structure, we performed circulardichroism (CD) experiments. Each of the three peptides tested had asignificant helical population in solution, approximating 12-14%.Furthermore, these peptides behave as monomers in solution, and anyhelical structure is intrinsic to the peptide, and not due toself-association into helical oligomers (Laskowitz et al., 2001). Ourresults are consistent with the possibility that the free peptides arecapable of adopting a helical conformation, which may be stabilized uponreceptor binding.

Clearly, the degree of helicity is not the only determinant ofbioactivity, as both the functional (apoE 133-149; apoE 136-149) andnon-functional (apoE 139-149) peptides had comparable helicity. Thus,the neuroprotective effects of these ApoE-mimetic peptides also appearto be dependent on the specific amino acid sequence and size. Inparticular, the 17 amino acid peptide derived from residues 133-149 ofthe apoE receptor-binding region completely blocked the toxicity of NMDAexposure, whereas the 14 amino peptide (apoE 136-149) had reducedefficacy, and the 11 amino acid sequence (apoE 139-149) lost allbiological activity in this regard. This suggests that residues 133-139are essential for bioactivity. Interestingly, this domain is identicalto that required for downregulation of glial activation (Laskowitz etal., 2001).

It is noteworthy that the peptides used in this study are derived fromthe receptor-binding region, and do not include residues 112 and 158,which are the polymorphic regions associated with the different humanapoE isoforms. Thus, although the current studies do not directlyaddress the association between apoE isoform and human disease, it iscertainly plausible that amino acid substitutions distant from thereceptor binding region may affect the conformation of this region, andsubsequent apoE-receptor interactions. For example, the substitution ofcysteine for arginine at position 158 significantly reduces the abilityof ApoE2 to bind the LDL receptor, even though this polymorphism liesoutside of the receptor binding region (Weisgraber et al., 1982).

Our results are in contrast to the reports of other groups, who haverecently suggested that the ApoE peptide fragments may cause neuronalinjury. For example, it has recently been demonstrated thatcarboxyl-terminal truncated forms of ApoE occur in the brains ofpatients with AD, presumably as a result of intracellular processing.These fragments are bioactive and are capable of interacting withcytoskeletal proteins to induce inclusions resembling neurofibrillarytangles in cultured neurons (Huang et al., 2001).

Our observation that peptides derived from the ApoE protein are capableof conferring neuroprotection against NMDA induced excitotoxicity isalso in contrast to other recent observations. The majority of thesestudies utilized tandem repeats derived from the receptor-binding regionof apoE. In particular, an eighteen amino acid peptide comprised oftandem repeats of residues 141-149 increases intracellular Ca²⁺ andregulates tau phosphorylation via two separate mechanisms: activation ofa cell surface Ca²⁺ channel, and release of internal Ca²⁺ stores via apertussis-toxin sensitive pathway (Wang and Gruenstein, 1997). Using apeptide comprised of a tandem repeat of residues 141-149, Tolar et al.demonstrated that exposure of primary hippocampal neurons to thispeptide induced neuronal cell death, an effect which was blocked bypreincubation with MK-801 (Tolar et al., 1999). These results predictthat exposure with the tandem repeat peptide would amplify NMDA-inducedexcitotoxicity by direct or indirect mechanisms. It is worth noting,however, that this tandem repeat peptide is substantively different thanthe peptides used in the current study. A recent report (Moulder et al.,1999) observed that neuronal cell death induced by this tandem repeatmay occur via a different mechanism than neuronal death induced by theholoprotein, suggesting that this tandem repeat may not be abiologically relevant model of the intact ApoE protein.

In summary, we report that small peptides derived from thereceptor-binding region of ApoE block the calcium influx andneurotoxicity associated with exposure of primary neocortical culturesto NMDA. The in vivo relevance of these findings is consistent with theclinical observations that ApoE appears to modulate neurologicalrecovery from ischemic and hemorrhagic stroke, as well as the globalcerebral hypoperfusion associated with cardiopulmonary bypass andpost-cardiac arrest resuscitation. Although the neuroprotection observedwith these peptides is greater than the modest benefit observedfollowing exposure to the intact ApoE protein, these observationssuggest that one mechanism by which endogenous ApoE may affect recoveryfrom ischemic injury is by protecting against glutamate excitotoxicity.The use of these ApoE-mimetic peptides should have therapeuticimplications, as well as provide further insight into the neurobiologyof this protein in brain subjected to an ischemic or traumatic insult.

EXAMPLE 13 Suppression of LPS-Induced TNF-a and IL-6 Production by ApoEMimetic Peptides

Septic shock is the most common cause of death in intensive care units,and represents a significant unmet medical challenge. Lipopolysaccharide(LPS) is a primary mediator of gram negative sepsis, and theupregulation of inflammatory cytokines induced by LPS plays an importantrole in mediating the systemic inflammatory response associated withsepsis. Intravenous administration of LPS is a common animal model ofgram negative septic shock, and replicates the clinically relevantsystemic inflammatory response. In particular, intravenousadministration of LPS causes early upregulation of TNFα and IL-6, whichare macrophage-derived cytokines that play an important role inmediating systemic inflammation.

We now demonstrate that injection of ApoE (133-149) suppresses serumlevels of TNFα and IL-6 following LPS administration. In these methods,14-16 week old male C57-BL6 mice were injected with LPS (11.25 μg in 150μl sterile isotonic saline, or 375 μg/kg) via the tail vein, and thenimmediately with vehicle (isotonic sterile saline) or ApoE (133-149) (ata dose of 200 μg, in 100 μl, or 6.6 mg/kg, prepared in isotonic saline).Serum samples were obtained in both LPS+vehicle and LPS+peptide groups(n=10 animals/group) at the following timepoints: time 0 (prior toinjection), 1 hour, 3 hour and 24 hours after injection. Blood wascollected by transcardial puncture, and allowed to clot for 30 minutes.Serum samples, obtained after centrifugation at 16,000 g for 5 minutes,were screened by solid phase ELISA for TNFα and IL-6. There were 20animals per time point.

At 1 hour post-injection, serum TNFα was significantly reduced as afunction of whether animals received LPS+vehicle or LPS+apoE peptide(FIG. 17A). At 3 hrs and at baseline, TNFα levels were not measurable.At 1 and 3 hours post-injection, serum IL-6 levels were significantlyreduced as a function of whether animals received LPS+vehicle orLPS+apoE peptide (FIG. 17B). At 24 hrs and at baseline, IL-6 levels werenot measurable. This data reveals that ApoE (133-149) suppresses TNFαand IL-6 production in the presence of LPS, and therefore suggests thatApoE mimetic peptides may have therapeutic potential in the clinicalsetting in patients with early sepsis.

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1. A method of treating or reducing the inflammation associated withsepsis in a subject in need thereof, comprising administering to thesubject a composition comprising an ApoE peptide, wherein said ApoEpeptide is a peptide of 18 to 50 amino acids comprising the sequence ofSEQ ID NO: 10, and wherein said composition is administered in an amountsufficient to reduce serum levels of inflammatory cytokines in thesubject as compared to that which would occur in the absence of saidcomposition.
 2. The method of claim 1, wherein said inflammatorycytokines include TNFα or IL-6.
 3. The method of claim 1, wherein saidcomposition is administered concurrently or sequentially with one ormore anti-inflammatory cytokines or monoclonal antibodies.
 4. The methodof claim 3, wherein said anti-inflammatory cytokines are selected fromthe group consisting of IL-10, transforming growth factor-beta,granulocyte colony-stimulating factor, IFN-phi, macrophage migrationinhibitory factor and high mobility group 1 protein.
 5. The method ofclaim 3, wherein said monoclonal antibodies are selected from the groupconsisting of antiendotoxin antibodies, anti-tumor necrosis factorantibodies, and anti-CD14 antibodies.
 6. A method of treating orreducing the inflammation associated with sepsis in a subject,comprising administering a composition comprising an ApoE peptide,wherein said ApoE peptide consists of the sequence of SEQ ID NO: 10, andwherein said composition is administered in an amount sufficient toreduce serum levels of inflammatory cytokines in the subject as comparedto that which would occur in the absence of the composition.